Clinical Lab Lectures combined.pdf

Yana Bocheva, MD, PhD Department of common medicine and clinical laboratory Medical University-Varna  Preanalysis, analysis, postanalysis  Reference ranges- creation, usage  Quality assurance in clinical laboratory  Er...

Yana Bocheva, MD, PhD Department of common medicine and clinical laboratory Medical University-Varna  Preanalysis, analysis, postanalysis  Reference ranges- creation, usage  Quality assurance in clinical laboratory  Errors in pre, post and analytical steps  Clinical laboratory measurements form the scientific basis upon which medical diagnosis and management of patients is established.  These results constitutes the largest section of medical record of patients, and laboratory examinations will only continue to grow in number as new procedures are offered and well established ones are ordered more frequently in the future.  Nowadays clinical laboratory is expected to provide methods and tests for early detection, diagnosis, prognosis and monitoring of patient‘s treatment.  Traditional areas of testing are: clinical chemistry, hematology, coagulation, microbiology, immunology, transfusion medicine.  Genetic testing for hereditary disease risk assessment is becoming a reality, beginning with individual disease testing that is expected to be followed by whole genome screening for a multitude of conditions.  Preanalysis refers to all the steps that take phase before a sample can be analyzed, such as ordering and sample collection  The analysis stage consists of the laboratory activities that in fact provide a result, such as running a sample on a automated analyzer or staining a blood smear.  Postanalysis comprises patient reporting and result interpretation.  Collectively all the interrelated laboratory steps in the testing process describe its workflow.  Series of studies identified that up to 75% of the so called “laboratory errors ” occur in the preanalytic phase, but the technologic advances and QC assurance procedures have reduced the number of analytic errors significantly.  Preanalitic factors include: 1. Patient related variables=Precollection variables. 2. Specimen collection and labelling techniques. 3. Specimen preservatives and anticoagulants. 4. Specimen transport and storage. Physiological factors Diurnal variation Cortisol, ACTH, Renin activity, aldosterone ,insulin , growth hormone, acid phosphatase, thyroxine, prolactin, iron, calcium , Hb, Ht, creatinine, potassium Exercise ↑free fatty acids, lactate, CK, AST, LDH ; activation of coagulation, fibrinolysis, platelets ↓LH, FSH, steroid hormones- long distance athletes Diet ↑rigth after meal- glucose ,, triglycerides potassium, ALP, 5- HIAA, leucocytes, iron, phosphates Vegeterians- ↓ LDLs, VLDLs, total lipids, Vit. B12, Meat- ↑BUN, Chronic alcohol abuse-↑HDL, GGT, MCV Obesity- ↑cholesterol, triglycerides, insulin, cortisol; ↓ testosterone in men  Physiological factors Stress ↑ ACTH, cortisol, catecholamines, total cholesterol, leucocytes, lactate, free fatty acids ↓HDL cholesterol Posture Upright- ↑ albumin, total protein, enzymes, Ca, bilirubin, cholesterol, triglycerides, Leu, RBC, PL, Bed rest- ↓Hb, Ht Age Newborn/bilirubin, RBC, Hb/, childhood to puberty/↑ALP, creatinine/, adult/↑cholesterol, triglycerides/, elderly adult/↓fT4, PTH, aldosterone, cortisol, testosterone, ↑FSH/. Gender Men- ↑Hb, Ht, RBC, iron, ALP, CK, calcium, ferritin, Mg  Common interferences In vivo Tobacco smoking: ↑Eo, Hb, RBC, MCV, WBC, lactate, insulin, growth hormone, Ig E ↓Neu, Mo, Vit B12, Ig A, Ig G, Ig M ↓sperm counts, motility; ↑ abnormal morphology of sperm cells In vitro Collection- associated variables/phlebotomy problems: Hemolysis- needles, tourniquet application, shaking or mixing tubes vigorously Icteric/ lipemic serums  Specimen collection errors Test order Incorrect test, wrong name/gender/age of the patient, wrong tube for analysis, Time of collection As soon as possible= stat TDM- Trough/peak level Rhythm of cortisol/ reproductive hormones Glucose tolerance test Specimen rejection Icterus/Hemolysis/Lipemia Clots In an anticoagulated specimen Nonfasting specimen Improper blood collection rube Short draws/wrong volume Improper transport Wrong ID/lack of ID on the tube Contaminated specimen  Open tubes, stored at room temperature  Storage in a place on direct sunlight or near heating device- hemolysis, enzyme denaturation  Leaving serum/plasma not removed from the cells- GGT↑ 27% for half an hour, creatinine↑110% in plasma and 60% in serum for 48 hours.  Serum or plasma-PTH has different RR;  Effect of froze/thaw cycles  Finger or Heel Skin Puncture  For infants younger than 12 months old, this is most usually the lateral or medial plantar heel surface.  For infants older than 12 months, children, and adults, the palmar surface of the last digit of the second, third, or fourth finger may be used.  The thumb and fifth finger must not be used, and the site of puncture must not be edematous or a previous puncture site because of accumulated tissue fluid.  Warm the puncture site with a warm, moist towel no hotter than 42° C;  Cleanse the puncture site with 70% aqueous isopropanol solution. Allow the area to dry.  Make the puncture with a sterile lancet or other skin-puncturing device, using a single deliberate motion nearly perpendicular to the skin surface. For a heel puncture, hold the heel with the forefinger at the arch and the thumb proximal to the puncture site at the ankle. If using a lancet, the blade should not be longer than 2 mm to avoid injury to the calcaneus (heel bone).  Discard the first drop of blood by wiping it away with a sterile pad. Regulate further blood flow by gentle thumb pressure. Do not milk the site, as this may cause hemolysis and introduce excess tissue fluid.  Collect the specimen in a suitable container by capillary action. Closed systems are available for collection of nonanticoagulated blood and with additives for whole blood analysis. Open-ended, narrow-bore disposable glass micropipets are most often used up to volumes of 200 µL. Both heparinized and nonheparinized micropipets are available. Use the appropriate anticoagulant for the test ordered. Mix the specimen as necessary.  Label the specimen container with date and time of collection and patient demographics.  Indicate in the report that test results are from skin puncture.  Arterial puncture technique- 0,05ml liquid heparin /1ml blood- recommended for blood gas analysis  Prepare the arterial blood gas syringe according to established procedures. The needle (18–20 gauge for brachial artery) should pierce the skin at an angle of approximately 45–60 degrees (90 degrees for femoral artery) in a slow and deliberate manner. Some degree of dorsiflexion of the wrist is necessary with the radial artery, for which a 23–25 gauge needle is used. The pulsations of blood into the syringe confirm that it will fill by arterial pressure alone.  After the required blood is collected, place dry gauze over the puncture site while quickly withdrawing the needle and the collection device.  Compress the puncture site quickly, expel air from the syringe, and activate the needle safety feature; discard into sharps container  Mix specimen thoroughly by gently rotating or inverting the syringe to ensure anticoagulation.  Place in ice water (or other coolant that will maintain a temperature of 1°–5° C) to minimize leukocyte consumption of oxygen.  Continue compression with a sterile gauze pad for a minimum of 3 to 5 minutes (timed). Apply an adhesive bandage.  Venous Puncture Technique  Verify that computer-printed labels match requisitions  Position the patient properly. Assemble equipment and supplies.  Apply a tourniquet and ask the patient to make a fist without vigorous hand pumping. Select a suitable vein for puncture.  Put on gloves with consideration of latex allergy for the patient.  Cleanse the venipuncture site with 70% isopropyl alcohol. Allow the area to dry.  Anchor the vein firmly.  Enter the skin with the needle at approximately a 30-degree angle or less to the arm, with the bevel of the needle up:  Follow the geography of the vein with the needle.  Insert the needle smoothly and fairly rapidly to minimize patient discomfort.  If using an evacuated system, as soon as the needle is in the vein, ease the tube forward in the holder as far as it will go, firmly securing the needle holder in place.  Release the tourniquet when blood begins to flow. Never withdraw the needle without removing the tourniquet.  Withdraw the needle, and then apply pressure to the site. Apply adhesive bandage strip over a cotton ball or gauze to adequately stop bleeding and to avoid a hematoma.  Mix and invert tubes with anticoagulant; do not shake the tubes. Check condition of the patient..  Label the tubes before leaving patient’s side.  TASKS  Sample introduction and transport to cuvette or dilution cup  Addition of reagent  Mixing of sample and reagent  Incubation  Detection  Calculations  Readout and result reporting ERRORS  Insufficient analytical specificity and sensitivity of the test  Incorrect calibration procedure  Failing automated equipment  Reagents  Laboratory staff  ERRORS  Wrong calculation  Results are reported to wrong patient  Incorrect interpretation  Alarms and Flags,  Flags for Problem Specimens  Flags for Specimens That Require Additional Analysis With Another Method  Flags for Problematic Results ASSESSMENT OF CLINICAL SIGNIFICANCE OF RESULTS  Critical Values  Reference Ranges  Definition of Reference Intervals  Factors That Influence Reference Ranges  Determination of Reference Ranges  Variability of Laboratory Results  Reference ranges can be used to separate normal from abnormal values.  Reference intervals are usually defined as the range of values into which 95% of non-diseased (“normal”) individuals will fall.  RR can be defined as “less than” or “greater than” a certain value-PSA.  Or can be defined as the measurement exceeding the 99th percentile of a reference control group” (Alpert, 2000)- cTr I , cTrT.  Laboratories frequently provide therapeutic target ranges that represent recommendations based on clinical trials and/or epidemiologic studies (Grundy, 2004)- lipids/ratios.  Drug measurements- therapeutic and toxic levels.  Factors, that influence RR:  Different laboratory metods( ALP, LDH, hormones ,Tu markers, Ig, infectious markers).  Different tubes/different anticoagulants(plasma/serum phenomenon) Determination of Reference Ranges  Laboratories are strongly encouraged to perform their own studies to establish reference ranges for all parameters, they report, usually by testing at least 120 samples from non- diseased individuals.  If this is not possible, the laboratory can verify a reference interval that it has previously established for a different method by transference (i.e., demonstrating that the new method yields identical results to the previous method).  If the parameter has not been previously tested for in the laboratory, the laboratory can verify another laboratory’s or the manufacturer’s reference interval (CLSI, 2008).  A critical value ( a panic or alert value) is a laboratory result that may represent a life-threatening situation that may not otherwise be detectable and therefore requires rapid reaction.  Regulations require that the critical value and the patient affected are read back by the health care provider to verify that the result was correctly communicated. The laboratory then has to document the communication of the critical value, the name and title of the caregiver who was notified, the time and date of notification, and the read- back by the care provider.  No universally accepted guidelines indicate which assays should have critical values, what the thresholds should be, whether critical values should be repeated before reporting, and what is an acceptable time from result availability to caregiver notification.  Inter-individual variation of laboratory results occurs because of factors specific to individual patients- CK, creatinine  Analytic variability is the result of assay imprecision and is expressed quantitatively as the coefficient of variation (CV) - glucose↓↓, creatinine- ↑.  Intra-individual variability is due to biologic changes that cause parameter levels to fluctuate over time- diurnal variations in cortisol levels, oestrogen levels that vary with the menstrual cycle, and seasonal variations of vitamin D.  Interindividual Intraindividual Index of CV, Analyte individual Method CV, % CV, % % Alanine aminotransferase 50.2 23.7 0.47 3.2 Albumin 8.9 2.8 0.31 3.4 Alkaline phosphatase 33.4 4.4 0.13 6.5 Apolipoprotein A 17.8 7.0 0.39 4.8 Apolipoprotein B 27.6 9.5 0.34 2.7 Aspartate aminotransferase 29.1 15.1 0.52 3.4 β-Carotene 67.4 24.2 0.36 7.4 Bicarbonate 13.3 11.0 0.83 2.4 Bilirubin, total 43.9 24.6 0.56 3.0 C-peptide 65.7 28.4 0.43 7.2 Calcium, ionized 3.6 2.4 0.67 1.4 Calcium, total 4.7 3.3 0.70 2.2 Chloride 3.1 1.9 0.61 1.0 Cholesterol, total 22.3 8.2 0.37 2.3 Creatinine 18.7 6.8 0.36 1.0 Creatinine, urine 61.3 43.0 0.70 2.2 Fibrinogen, plasma 25.6 16.2 0.63 3.9 Folate 64.3 22.6 0.35 3.6 γ-Glutamyl transferase 59.8 16.2 0.27 1.7 Glucose, plasma 12.5 8.3 0.66 1.7  Diagnostic Accuracy  Reference range can be used to separate normal from abnormal values, but clinically validated thresholds are commonly used for disease classification. The RR for glucose in a laboratory may be 3,5- 5,6 mmol/l, but the clinically established threshold to classify a patient as diabetic is 7,2mmol/l. This means that a patient’s result can be outside the reference range without meeting the threshold for diabetes.  A test with perfect diagnostic accuracy could determine the presence or absence of disease with certainty, and the established cutoff point would perfectly separate diseased from not diseased populations.  The diagnostic accuracy of a test is determined by comparing the test’s ability to discern true disease from nondisease. Patients correctly classified as abnormal are called true-positives (TPs) and those correctly classified as normal are called true-negatives (TNs).  These true results are the nonoverlapping areas of the two patient distributions. False results occur because the two populations overlap (i.e., because a test cannot completely discriminate all abnormal patients from normal ones).  Patients incorrectly classified as normal are false-negatives (FNs), and those incorrectly classified as abnormal are false-positives (FPs).  False results are produced when an analyte has two relevant cutoffs (e.g., thyroid-stimulating hormone), with overlapping populations at both the low end and the high end.   Sensitivity and specificity are measures of the diagnostic accuracy of a test.  Sensitivity is the ability of a test to detect disease and is expressed as the proportion of persons with disease in whom the test is positive.  A test that is 90% sensitive will give positive results in 90% of diseased patients (TP) and negative results in 10% of diseased patients (FN). 82% of patients having fasting glucose 82% sensitivity of fasting level>104 milligram/dL are diagnosed glucose levels diabetes, 18% of diabetes patients will be false negative (< 104 mg/dl)  Specificity is the ability to detect the absence of disease and is expressed as the proportion of persons without disease in whom the test is negative.  A test that is 90% specific will give negative results in 90% of patients without disease (TN) and positive results in 10% of patients without disease (FP).  A test with a higher sensitivity identifies a greater proportion of persons with disease, and a test with a higher specificity excludes a greater proportion of persons without disease. 90% of patients with PSA < 4 ng/ml are classified PSA-90% specificity non-cancer patients (true negative), 10% of non- cancer patients are false positive( >4ng/ml PSA)   The predictive value of a positive test (PPV) may be understood as the probability that a positive test indicates disease. It is the proportion of persons with a positive test who truly have the disease.  The predictive value of a negative test (NPV) is the probability that a negative D-dimer tested in a group of test indicates absence of patients with clinical symptoms of disease. It is the proportion deep venous thrombosis. of persons with a negative test who are truly without disease. Sensitivity= 0.34=34% Specificity=1.00=100%= =PPV NPV  Internal Quality control  Belk и Sundermann,  External Quality control 1947 (EQAS=Proficiency testing)  Levy и Jennings, 1951 (ВКК)  Wooten и King, 1953 (ВОК)  Total Quality Management  Акредитация  Internal quality control at the chemical analytical laboratory involves a continuous, critical evaluation of the laboratory’s own analytical methods and working routines. The control encompasses the analytical process starting with the sample entering the laboratory and ending with the analytical report.  The most important tool in this quality control is the use of control charts. The basis is that the laboratory runs control samples together with the test samples. The control values are plotted in a control chart. In this way it is possible to demonstrate that the measurement procedure performs within given limits. If the control value is outside the limits, no analytical results are reported and remedial actions have to be taken to identify the sources of error, and to remove such errors.  Random error=imprecision is either positive or negative and its magnitude and direction can not be predicted (pipetting, thawing & frozing of the control material).  Systematic error=inaccuracy is always in one direction and displays the mean of the distribution from its regular place(target). They cause all the test results to be either high or law. Usually due to calibration or reagent.  Proficiency testing (or external quality assessment) consists of evaluation of method performance by comparison of results versus those of other laboratories for the same set of samples.  PT providers circulate a set of samples among a group of laboratories. Each laboratory includes the PT samples along with patient samples in the usual assay process. Results for the PT samples are reported to the PT provider for evaluation.  PT allows a laboratory to verify that its results are consistent with those of other laboratories using the same or similar methods for an analyte, and to verify that it is using a method in conformance with the manufacturer’s specifications. Typical evaluation report sent to a participating laboratory. Clin lab mid 21.3/2024 Poor(2) Attended= certificate of semester test participation Clin lab exam 1.06.2024 Very good (5) Passed (nice summer vacation)= certificate of quality  Yana Bocheva, MD, PhD Department of common medicine and clinical laboratory Medical University-Varna  Indications for analysis and reference ranges of the basic haematological parameters  Red blood cell count- methods of determination, reference ranges, indications for determination, interfering factors.  Haemoglobin- methods of determination, indications for analysis and reference ranges  Haematocrit- indications for analysis and reference ranges  Erythrocyte indices – MCV, MCH, MCHC, RDW- methods of determination, reference ranges, indications for determination, clinical significance.  Bone marrow examination- indication, specimen and technique, rules for preparation and examination  Investigation and classification of anaemia  Clinical case  Blood consists of plasma, a pale-yellow, coagulable fluid, in which various types of blood cells are suspended. The cells comprise erythrocytes, granulocytes, monocytes, lymphocytes and platelets.  Every study and clinical examination of a patient starts with full blood count(FBC):  An usual parameter for monitoring hematologic diseases.  A basic part for health screening according to WHO.  Hemoglobin (Hb), the main component of the red blood cell (RBC), is a conjugated protein that serves as the vehicle for the transportation of oxygen (O2) and carbon dioxide.  A molecule of Hb consists of two pairs of polypeptide chains (“globins”) and four prosthetic heme groups, each containing one atom of ferrous iron.  Hemiglobincyanide Method-Hemoglobin is oxidized to cyanmethemoglobin by the addition of cyanide,and the cyanmethemoglobin is then determined spectrophotometrically at 540 nm by the automated counter.  Hb F (α2γ2) - 0.2-1%. The main type in fetuses and newborns. Till the 18 th month it is changed by Hb A. Its concentration is raised in adults with sickle cell anemia and thalassemia.  Hb A (α2β2) - 95-98%. This is the main part of the hemoglobin in adults. Its levels lower in some hemoglobinopathies.  Hb A2 (α2δ2) -2- 3.5%. A normal type of hemoglobin in low concentrations in adults.  Methemoglobin, Sulfhemoglobin, Carboxyhemoglobin- acquired not functional forms of hemoglobin. Beta thalassemia trait Hb E heterozygote Hemoglobin Sickle cell C disease disease Beta thalassemia Beta thalassemia trait homozygote Hb E β Hb E β thalassemia thalassemia  Methemoglobin- Congenital methemoglobinemia due to cytochrome b5 reductase deficiency is very rare. Most cases of methemoglobinemia are classified as secondary or acquired, due mainly to exposure to drugs and chemicals. Chemicals or drugs that directly lead to the conversion of some or all of the four iron species from the reduced ferrous (Fe2+) state to the oxidized ferric (Fe3+) state are nitrites, nitrates(well water consumption), chlorates, and quinones, benzocaine and prilocaine.  Methemoglobinemia is a clinical diagnosis based on history and presenting symptoms, including hypoxemia refractory to supplemental oxygen and the presence of chocolate-colored blood. The diagnosis is confirmed by arterial or venous blood gas with co- oximetry, which will determine the methemoglobin concentration and percentage  Treatment of methemoglobinemia includes:  removal of the inciting agent  treatment with the antidote, methylene blue (tetramethylthionine chloride).  high flow oxygen delivered Aim: to increase oxygen delivery to tissues and enhances the natural degradation of methemoglobin. NB G6PD deficient patients and NADPH-MetHb reductase deficiency! Additional options for treatment: ascorbic acid, exchange transfusion, hyperbaric oxygen therapy  Sulfhemoglobin- SHb has been reported in patients receiving treatment with sulfonamides or aromatic amine drugs (phenacetin, acetanilid), as well as in patients with severe constipation, in cases of bacteremia due to Clostridium perfringens, and in a condition known as enterogenous cyanosis.  Carboxyhemoglobin-Acute CO poisoning is well known. It produces tissue hypoxia as a result of decreased O2 transport. Chronic poisoning, a result of prolonged exposure to small amounts of CO, is less well recognized but is of increasing importance. The chief sources of the gas are gasoline motors, illuminating gas, gas heaters, defective stoves, and the smoking of tobacco.  The Hct of a sample of blood is the ratio of the volume of erythrocytes to that of the whole blood. It may be expressed as a percentage (conventional) or as a decimal fraction (SI units).  The Hct may be measured directly by centrifugation with macro methods or micro methods, or calculated by the hematological analyzers(Hct = MCV х Erys).  In blood kept at room temperature, swelling of erythrocytes between 6 and 24 hours raises Hct and MCV. Cell counts and indices are stable for 24 hours at 4° C 5 min./16 000 G The length of thered cell column alone is divided to the length of the blood column, including the plasma (2.1 см/5,9) x100= Hct% =35%  Typical reference values for adult males are 0.41–0.51, and for females, 0.36–0.45. A value below an individual’s normal value or below the reference interval for age and sex indicates anemia, and a higher value, polycythemia.  The Hct reflects the concentration of red cells—not the total red cell mass.  The Hct is low in hydremia of pregnancy, but the total number of circulating red cells is not reduced. The Hct may be normal or even high in shock accompanied by hemoconcentration, although the total red cell mass may be decreased considerably owing to blood loss.  The Hct is unreliable as an estimate of anemia immediately after loss of blood or immediately following transfusions.  Wintrobe introduced calculations for determining the size, content, and Hb concentration of red cells; these erythrocyte indices have been useful in the morphologic characterization of anemias. They may be calculated from the red cell count, Hb concentration, and Hct. Mean Cell Volume  The MCV, the average volume of red cells, is calculated from the Hct and the red cell count. MCV = Hct × 1000/RBC (in millions per µL), expressed in femtoliters or cubic micrometers. If the Hct = 0.45 and the red cell count = 5 × 1012/L, 1 L will contain 5 × 1012 red cells, which occupy a volume of 0.45 L. MCV = Hct/Erys ( fl )  RR (82-96) fl  Mean Cell Hemoglobin  The MCH is the content (weight) of Hb of the average red cell; it is calculated from the Hb concentration and the red cell count. MCH = Hb/Erys ( pg )  The value is expressed in picograms. If the Hb = 15 g/dL and the red cell count is 5 × 1012/L, 1 L contains 150 g of Hb distributed in 5 × 1012 cells.  RR(27-33) pg  Mean Cell Hemoglobin Concentration  The mean cell hemoglobin concentration (MCHC) is the average concentration of Hb in a given volume of packed red cells. It is calculated from the Hb concentration and the Hct. MCHC = Hb/Hct ( g/l ) MCHC = MCH/MCV  RR(300-360) g/l Meaning/interpretation MCH versus MCHC  Red Cell Distribution Width (RDW)  Modern analyzers also record the red cell distribution width (cell volume distribution). In normal erythrocyte morphology, this correlates with the Price-Jones curve for the cell diameter distribution. Discrepancies are used diagnostically and indicate the presence of microspherocytes (smaller cells with lighter central pallor).  RR(11.5% - 14.5%)  10 HBG 9 g/dl 12-16 g/dl MCV 89 fl 80-99 fl MCH 29 pg 27-32 pg MCHC 311 320-350 RDW 23% 11-15% Normocytic normochromic anemia RDW- Normal Pseudonormocytic normochromic anemia RDW- high  Manual Methods Except for some platelet counts and low leukocyte counts, the hemocytometer is no longer used for routine blood cell counting. Any cell counting procedure includes four steps: dilution of the blood; sampling of the diluted suspension into a measured volume; and counting of the cells in that volume, calculation. dilution of the blood dilution of the blood  Counting of the cells in that volume and calculation RBC = А х 200 х 4 000 = sampling of the diluted suspension =А х 10 000 / 80 in µl into a measured volume Other Methods for Manual Blood Cell Counting Романовски-Giemsa New Methylene Blue СДО 2016  The multichannel instruments used in the modern laboratory for performing cell counts are based on the principles of electrical impedance, light scattering, radiofrequency conductivity, and/or cytochemistry.  Combining hematology instrumentation with laboratory automation allows for reduction in pre-analytic and post-analytic variables and positive identification of samples for processing and analysis, storage, and retrieval.  Cells passing through an aperture through which a current is flowing cause changes in electrical resistance that are counted as voltage pulses  An accurately diluted suspension of blood (CS) is made in an isotonic conductive solution that preserves the cell shape  Each cell that passes through the aperture displaces an equal volume of conductive fluid, increasing the electrical resistance and creating a voltage pulse, because its resistance is much greater than that of the conductive solution. The pulses, which are proportional in height to the volume of the cells, are counted. This is the Coulter principle.  Conductivity is determined  In the electro-optical using a high-frequency analyzers a light-sensitive electromagnetic probe that detector measures light provides information on the scattering. The size of the cells’ internal constituents pulse detected is (chemical composition, proportional to the size of nuclear characteristics, and the particle (WBC, RBC, or granular constituents) by platelet). permeating the lipid layer  Although the precision of the of a cell’s membrane. instruments employing optical methods is equivalent Conductivity is especially to that of systems utilizing helpful in differentiating electrical impedance, some between cells of like size systems use a combination of such as small lymphocytes the two methods to supply and basophils. an internal comparison. Radiofrequency Light Scattering Conductivity  A method unique to the Siemens automated hematology series (Siemens Healthcare Diagnostics, Deerfield, Ill.) is the use of a cytochemical reaction to determine the peroxidase activity of white blood cells. The mean peroxidase index (MPXI), a measure of neutrophil-staining intensity, is determined for each specimen. The relative positivity seen in neutrophils, eosinophils, and monocytes is used in conjunction with data derived from light scatter to determine the WBC differential Cytochemistry  Erythrocyte sedimentation rate (ESR) is a useful but nonspecific marker of underlying inflammation. Recently, high-sensitivity C-reactive protein and other inflammatory markers have been used to detect or monitor disease, particularly cardiovascular disease and metabolic syndrome. When well-mixed venous blood is placed in a vertical tube, erythrocytes will tend to fall toward the bottom. The length of fall of the top of the column of erythrocytes over a given interval of time is called the ESR.  Three stages can be observed: (1) In the initial 10 minutes, little sedimentation occurs; (2) for about 40 minutes, settling occurs at a constant rate; and (3) sedimentation slows in the final 10 minutes as cells pack at the bottom of the tube.   1 = Normal ESR  2 = Normal ESR = hemolysis  3 =Reticulocytosis  4 = Leucocytosis/leukemia  5 = Lipemia  6 = Icterus  7 = 0 ESR in polycytemia vera  8 = Multiple Myeloma Panchenko method Case 1-8  Alternative Methods and Technologies to Measure ESR Micro-ESR method has greater utility in Westergren Method pediatric patients  Moderate elevations are common in active inflammatory disease such as rheumatoid arthritis, chronic infection, collagen disease, and neoplastic disease.  The ESR can be useful in monitoring disease activity. It is simpler than measurement of serum proteins, which has tended to replace ESR.  Because the test is often normal in patients with neoplasm, connective tissue disease, and infection, a normal ESR cannot be used to exclude these diagnostic possibilities.  In patients with known cancer, however, when the value exceeds 100 mm/hour, metastases are usually present.  The ESR is of little value in screening asymptomatic patients for disease; history and physical examination will usually disclose the cause of an elevated ESR.  The ESR is useful and is indicated in establishing the diagnosis and in monitoring polymyalgia rheumatica and temporal arteritis, where the rate typically exceeds 90 mm/hour.  Emergency physicians continue to use the ESR in evaluating temporal arteritis, septic arthritis, pelvic inflammatory disease, and appendicitis.  The bone marrow examination provides a semiquantitative and qualitative assessment of the state of hematopoiesis and aids in the diagnosis of several hereditary and acquired benign and malignant diseases.  Marrow aspiration and biopsy can be carried out as an office procedure on ambulatory patients with minimal risk. It compares favorably with ordinary venipuncture and is less traumatizing than a lumbar puncture.  As for any other special procedure, however, the clinical indications for marrow examination should be clear.  Without exception, the peripheral blood should be examined carefully first. It is a relatively uncommon circumstance to find hematologic disease in the bone marrow without evidence of it in the peripheral blood.  It is estimated that the weight of the marrow in the adult is 1300–1500 g.  Diagnostic indications ◦ Abnormal FBC and peripheral blood stain ◦ Anemia ◦ Neutropenia, thrombocytopenia, or pancytopenia ◦ Paraprotein / M spike/ ◦ Non clear septic conditions- AIDS, Tuberculosis, Splenomegalia  Staging ◦ Lymphoma  Therapy response ◦ Leukemia, Multiple myeloma, Lymphoma  Marrow films should be stained with Romanowsky’s stain (e.g., Wright-Giemsa) in a manner similar to that used for blood films.  A longer staining time may be necessary for marrows with greater cellularity. Several special stains may be performed on peripheral blood smears, bone marrow aspirate, and touch imprint smears and bone marrow biopsy sections, besides the usual Romanowsky’s stains. These include cytochemical stains (myeloperoxidase, Sudan black B, naphthol As-D chloroacetate esterase, nonspecific esterases, acid phosphatases, leukocyte alkaline phosphatase, periodic acid–Schiff stain, toluidine blue, and iron stain) and immunocytochemical stains, depending on the disease.  Peripheral Blood  Cellularity of the Marrow  Distribution of Cells Maturation  Presence of Rare Cell Types or  Abnormal Cells  Evaluation of the Biopsy Specimen The summary of the marrow report includes an estimate of cellularity, an estimate of the number of megakaryocytes, the M/E ratio, statements about any cytologic or maturation abnormalities, an estimate of the storage iron and proportion of sideroblasts, and statements about any other abnormal findings present  Anemia is considered to be present if the hemoglobin (Hb) concentration or the hematocrit (Hct) is below the lower limit of the 95% reference interval for the individual’s age, sex, and geographic location.  This means that 2.5% of normal individuals will be classified as anemic.  Anemia may be absolute, when red blood cell mass is decreased, or relative, when associated with a higher plasma volume.  Causes of absolute anemia fall into two major pathophysiologic categories: impaired red cell production and increased erythrocyte destruction or loss in excess of the ability of the marrow to replace these losses.  Several authors have included posthemorrhagic anemia in the latter category.  Anemia may be classified by red cell morphology as macrocytic, normocytic, or microcytic—an approach that is useful in differential diagnosis.  In general, the anemic patient complains of easy fatigability and dyspnea on exertion, and often of faintness, vertigo, palpitations, and headache. The more common physical findings are pallor, a rapid bounding pulse, low blood pressure, slight fever, some dependent edema, and systolic murmurs. In addition to these general signs and symptoms, certain clinical findings are characteristic of the specific type of anemia.  Clinical signs and symptoms result from diminished delivery of oxygen.  When anemia develops slowly in a patient who is not otherwise severely ill, Hb concentrations as low as 60 g/L may develop without producing any discomfort or physical signs, as long as the patient is at rest.  ACUTE Chronic blood loss  When iron loss exceeds iron intake for a time long enough to deplete the body’s iron stores, insufficient iron is available for normal Hb production. When well developed, iron deficiency is characterized by a hypochromic microcytic anemia.  Iron deficiency is probably the most common cause of anemia on the planet, affecting at least one third of the world’s population.  Children between the ages of 6 and 24 months are particularly susceptible. It is caused by insufficient dietary iron to meet the needs of rapid growth. After the first 4–6 months of life, the iron stores present from birth have been exhausted, and the infant depends on dietary iron.  In a study in the United States, iron deficiency anemia was reported in 3% of toddlers aged 1–2 years and in 2%–5% of adolescent girls and women of childbearing age  Iron deficiency anemia occurs after total gastrectomy or even subtotal gastrectomy. Prolonged treatment of peptic ulcer and acid reflux by H2 blockers and acid pump blockers may cause defective iron absorption.  Except for the sprue syndrome, causes of malabsorption of iron are extremely rare.  If an adult male had absolutely no iron intake or absorption (which would be extremely unlikely), his body iron stores of 1000 mg would last for 3–4 years before he would even begin to become iron deficient. Therefore, almost all cases of iron deficiency in adult males are due to chronic blood loss. Hemorrhagic lesions, such as benign and malignant tumors, chronic ingestion of some medications, and helminthic infections are common causes of iron deficiency in males and postmenopausal females.  In early iron deficiency status, the stained blood film shows normochromic normocytic erythrocytes.  In later stages, the picture is one of microcytosis, anisocytosis, poikilocytosis (including elliptical and elongated cells), and hypochromia, poikilocytes, particularly elongated hypochromic elliptocytes (pencil cells).  Anisocytosis may be identified by automated blood counters as increased red cell distribution width (RDW). This finding, however, is not specific for iron deficiency anemia.  Reticulocytes are usually decreased in absolute numbers, except following iron therapy.  The mean corpuscular volume (MCV) is low, and Hb and Hct are relatively lower than the erythrocyte count. Osmotic fragility may be decreased.  The leukocyte count is normal or slightly lowered. Granulocytopenia and a small number of hypersegmented neutrophils may be present.  Platelets may be increased, whether the lack of iron is due to blood loss.  Serum Iron  The reference interval is 50–160 µg/dL (9–29 µmol/L) in adults. The level is lower in iron deficiency and in infection and anemia of chronic disease.  Serum (Total) Iron-Binding Capacity  The reference interval for adults is 250–400 µg/dL (45–72 µmol/L). In iron deficiency anemia, the serum TIBC is increased. It is normal or decreased in the anemia of chronic disease. If chronic infection coexists with chronic blood loss, the TIBC may not be increased, even though the patient is iron deficient.  Percent Saturation of TIBC  The ratio of serum iron to TIBC is the percent saturation of the TIBC. Normally, this is 20%–55%; values below 15% indicate iron- deficient erythropoiesis.  Serum Ferritin is usually low. NB acute phase reactant  Iron deficiency anemia is associated with increased serum levels of TfRs.  The first principle in therapy of iron deficiency anemia is that the underlying cause can be identified and corrected.  Ferrous iron is given orally—at about 200 mg/day—in three doses between meals. This will provide 40–60 mg of absorbed iron per day, which, with the iron produced by turnover of senescent red cells, will be sufficient to increase production to two or three times normal.  The reticulocyte count will reach a maximum at 5–10 days, then will gradually decrease toward normal.  Monitoring the Hb is best; Hb should increase by 0.1–0.2 g/dL/day after the fifth day, and by at least 2 g/dL for each of the subsequent 3 weeks.  After the Hb has returned to normal, iron therapy should be continued for at least 2 months to replenish storage iron.  Patients refractory to treatment need to be investigated for continued underlying diseases, particularly chronic gastritis and Helicobacter pylori gastritis.  Macrocytic anemias that are not megaloblastic may be due to early release of erythrocytes from the marrow, so-called shift reticulocytes. This may occur in response to acute blood loss, hemolysis, bone marrow infiltration, and high levels of EPO associated with bone marrow failure diseases such as aplastic anemia, refractory anemia, and Diamond-Blackfan anemia. Nonmegaloblastic macrocytosis is also found in hypothyroidism, in individuals with excessive alcohol intake, and in liver disease.  Macrocytic anemias associated with megaloblastosis differ from nonmegaloblastic macrocytic anemia in that macroovalocytes and giant hypersegmented neutrophils are present in the blood.  Elevated MCV, anisocytosis and poikilocytosis.  Pancytopenia is the rule  Macrocytes and dacrocytes are common.  Basophilic stippling, multiple Howell-Jolly bodies, nucleated red cells with karyorrhexis, and even megaloblasts may be seen.  Leukopenia is present. Granulocytes have increased numbers of lobes, presumably as a result of abnormal nuclear maturation. Five lobes in more than 5% of the neutrophils constitute hypersegmentation as do any neutrophils with six or more lobes.  Thrombocytopenia is usually encountered and on rare occasions is sufficiently severe to be responsible for bleeding.  Megaloblastic anemia is characterized by enlargement of all rapidly proliferating cells of the body, including marrow cells. The major abnormality is the diminished capacity for deoxyribonucleic acid (DNA) synthesis.  With such deficiency, the cells have both a prolonged intermitotic resting phase and a block early in mitosis.The nuclei undergo karyorrhexis readily, and multiple Howell-Jolly bodies may be present. Usually more cells analogous to the pronormoblast and the basophilic normoblast are noted (i.e., promegaloblast and basophilic megaloblast) than are seen in normal erythropoiesis. This has sometimes been termed maturation arrest, or nuclear- cytoplasmic asynchrony.  The granulocytic series, the cells are larger, with retarded nuclear maturation.  The giant metamyelocyte is the most characteristic of the abnormal granulocytes. Megakaryocytes, too, are large and have separated nuclear lobes or nuclear fragments.  The bone marrow is hyperplastic.  Although the true prevalence of cobalamin deficiency in the general population is unknown, it increases with age. Approximately 15% of adults older than 65 years have laboratory findings of vitamin B12 deficiency. This prevalence may be attributed to the high frequency of hypochlorhydria of 25%–50%, which has been reported in the elderly population.  Anti–parietal cell antibodies  Anti–intrinsic factor antibodies occur in the serum, saliva, and gastric juice of about 75% of patients with PA.  Folic Acid Deficiency  With the patient on a diet low in cobalamin and folate, a parenteral physiologic dose of cobalamin (10 µg/day) is given. Optimal hematologic response indicates deficiency and consists of reticulocytosis beginning on the third or fourth day, reaching a peak on the seventh day. Erythropoiesis becomes normoblastic by 2 days, and leukopoiesis becomes normal by 12 to 14 days. Within a week, leukocyte and platelet counts have returned to normal, and the Hb concentration begins to rise.  PA is treated parenterally with 1000 µg of cyanocobalamin daily for 1 week, twice weekly for the second week, once weekly for 4 weeks, then monthly for the lifetime of the patient.  In folate deficiency, oral therapy is generally used at a dosage of 1–2 mg/day. Cobalamin deficiency must be excluded and corrected if present, to avoid the occurrence of neuropathies of cobalamin deficiency. Supplemental dietary folic acid during pregnancy is reported to reduce the incidence of neural tube defects in the baby  ACD designates an anemia syndrome typically found in patients with chronic infections or inflammatory or neoplastic disorders; it is characterized by reduced reticulocyte response accompanied by low serum iron, despite adequate iron stores. It is also termed anemia of chronic disorder, inflammation, or cytokine response. ACD occurs in approximately 50% of hospitalized patients, as identified by laboratory studies. The frequency is higher in the elderly population. ACDs have also been observed in acute trauma and critical care patients.  Erythrocytes are usually normocytic and normochromic, although in 20% of patients, the anemia is microcytic and hypochromic. Anisocytosis and poikilocytosis are slight. The reticulocyte count usually is not elevated. Leukocytes and platelets are not distinctively altered, except by the causative disease.  The marrow is normocellular or minimally hypocellular or hypercellular, and the cell distribution is not greatly disturbed. The normoblasts may have frayed hypochromic cytoplasm, and the appearance of Hb in the cells may be delayed (as in iron deficiency anemia). Sideroblasts are decreased, but storage of iron is normal or increased.  The serum iron concentration is characteristically decreased, the TIBC is decreased or normal (in contrast to iron deficiency anemia, in which the TIBC is elevated), and the percent saturation is decreased. Erythrocyte protoporphyrin and serum ferritin are elevated.  EPO levels, although above normal, have been disproportionate to the degree of anemia, indicating relative EPO deficiency in ACD.  Recently, hepcidin has been shown to be elevated in ACD through induction by IL-6 and is considered an acute phase reactant. As mentioned earlier, hepcidin interferes with the release of intracellular iron.  The anemia usually fails to respond to iron therapy.  However, patients treated with EPO have shown improvement.  ADVIA 2120 Iron Deficiency Anemia %MICRO / %HYPO < 0,9 Hypo: %hypo RBC≥4%  Micro: %micro RBC≥ 2.5% Restricted © Siemens AG 2015 All rights reserved. Page 63 2015-09-15 & 2015-09-16 Philippe RENARD / H CX CS RSC EMEA4 2 2  ADVIA 2120 Heterozygous ß thalassemia %MICRO / %HYPO > 0,9 Restricted © Siemens AG 2015 All rights reserved. Page 64 2015-09-15 & 2015-09-16 Philippe RENARD / H CX CS RSC EMEA4 2 2  ADVIA 2120 Double deficiency anemia type Restricted © Siemens AG 2015 All rights reserved. Page 65 2015-09-15 & 2015-09-16 Philippe RENARD / H CX CS RSC EMEA4 2 2  ADVIA 2120 Megaloblastic anemia Restricted © Siemens AG 2015 All rights reserved. Page 66 2015-09-15 & 2015-09-16 Philippe RENARD / H CX CS RSC EMEA4 2 2  ADVIA 2120 Non-megaloblastic macrocytic anemia HYPER: %Hyper RBC≥4% MACRO: %Macro RBC≥2.5% Normal RDW%  High MCV и MCH Restricted © Siemens AG 2015 All rights reserved. Page 67 2015-09-15 & 2015-09-16 Philippe RENARD / H CX CS RSC EMEA4 2 2  ADVIA 2120 Post transfusion changes High RDW Two single populations RBC in the cytogram Restricted © Siemens AG 2015 All rights reserved. Page 68 2015-09-15 & 2015-09-16 Philippe RENARD / H CX CS RSC EMEA4 2 2 Thank you for your attention! Laboratory diagnosis of the neoplastic and nonneoplastic leukocytic disorders Y.Bocheva, MD, PhD  Hematopoesis  In postnatal life in humans, erythrocytes, granulocytes, monocytes, and platelets are normally produced only in the bone marrow. Lymphocytes are produced in the secondary lymphoid organs, as well as in the bone marrow and thymus gland.  Hematopoesis In the standard model of hematopoiesis, multipotent progenitors give rise to common myeloid progenitors and common lymphoid progenitors. Common myeloid progenitors give rise to granulocyte/macrophage lineage–restricted progenitors and megakaryocyte/erythrocyte lineage–restricted progenitors. Common lymphoid progenitors give rise to B lymphocytes, T lymphocytes, and natural killer cells. The capacity for self-renewal is progressively lost, and terminally differentiated cells cannot divide Hematopoietic Growth Factors  Soluble or membrane-bound biochemical factors contributing to control of hematopoiesis include hematopoietic growth factors and interleukins(3,5,6,9,11).  They regulate the proliferation and differentiation of hematopoietic precursor cells and facilitate the function of mature blood cells. Hematopoietic growth factors may act locally near the site at which they are produced, or they may circulate in the blood. They act at low concentrations, are produced by many different types of cells, and usually affect more than one lineage.  Granulocyte/macrophage colony–stimulating factor, Granulocyte colony- stimulating factor, Monocyte/macrophage colony-stimulating factor, Erythropoietin, Thrombopoetin, Stem cell factor.  Laboratory assays are available for most hematopoietic growth factors and cytokines. Neutrophils  Neutrophils  Mature neutrophils express CD13, CD15, CD16, and CD11b, but lose HLA-DR and CD33.  The mature human neutrophil has twice as many specific granules as azurophilic granules.  From the time of differentiation into a myeloblast, through about five mitotic divisions (three of which occur at the myelocyte stage), it takes about 14 days until the progeny of that cell reach the blood. The last 6–7 days are spent in the maturation and storage pool.  If not utilized in an inflammatory exudate, neutrophils leave the body within a few days via secretions in bronchi, saliva, gastrointestinal tract, and urine, or they are destroyed by the mononuclear phagocyte system.  Basic function- bactericidal activity. Myeloblast-Promyelocyte-Myelocyte- Metamyelocyte-Band-Segmented Neu  Neutrophilia  Neutrophilic leukocytosis or neutrophilia refers to an absolute concentration of neutrophils in the blood above normal for age. The normal reference interval (established for each laboratory separately) is approximately 2–7.0 × 103/µL for adults, with a slightly wider range (1.0–8.5 × 103/µL) in The primary factors influencing young children. The neutrophilias the neutrophil count are (1) the rate of inflow of cells from the are acquired disorders; none is BM; (2) the proportion of inherited neutrophils in the marginal granulocyte pool and the circulating granulocyte pool of the and (3) the rate of outflow of neutrophils from the blood  Neutrophilia  Physiologic leukocytosis is produced by factors or situations that are not related to underlying tissue pathology. Severe exercise, hypoxia, stress, or injection of epinephrine.  Neutrophilia may be produced by corticosteroids, which increase the release of neutrophils from the marrow, decrease the egress of neutrophils from the blood, and increase demargination.  Neutrophilia can also be seen with gastrointestinal and hepatic tumors, renal cell carcinoma, and metastatic disease.  Neutrophilia  Pathologic leukocytosis is an increased WBC count that occurs as a result of disease, usually as a response to tissue damage. This leukocytosis is most often a neutrophilia.  In acute infection, increased margination of neutrophils and outflow from blood to tissues would lead to neutropenia were there not a flow of neutrophils from the marrow storage compartment into the blood. Because the latter overcompensates, the result is a neutrophilia. Usually, production and storage compartments then increase in the marrow. In these instances, the marrow will show granulocytic hyperplasia (increased myeloid to erythroid [M : E] ratio and increased cellularity), with maturation intact. An increase in immature peripheral blood granulocytes is usually present, often termed “shift to the left.”  Neutropenia  Neutropenia is a reduction in the absolute neutrophil count (ANC) below ≈1.5–2 × 109/L for white adults and below ≈1.2–1.3 × 109/L for black adults. Remember that the ANC is the product of the WBC count and the percentage of neutrophils and bands that have been enumerated in the WBC differential count.  The term agranulocytosis has been used for severe neutropenia, usually 40% Ly.  In contrast to infectious mononucleosis, atypical lymphocytes are uncommon.  A chronic form of infectious lymphocytosis also occurs in children. The leukocyte count is 10–25 × 109/L, with 60%–80% lymphocytes of normal appearance. Slight eosinophilia, monocytosis, and plasmacytosis are also present. As a rule, children have enlargement of tonsils, lymph nodes, and spleen and a history of recurrent upper respiratory infection. The marrow shows no abnormalities.  Infectious Mononucleosis and Epstein-Barr Virus Infection  Infectious mononucleosis (IM) is usually a self-limited infectious disease characterized by sore throat, prolonged malaise, atypical lymphocytosis with the presence of large transformed lymphocytes , lymphadenopathy (most often posterior cervical), and often splenomegaly. In immunocompromised patients, EBV is associated with benign B cell hyperplasia, malignant lymphoma, and posttransplantation lymphoproliferative disease.  IM is a disorder that occurs secondary to infection with EBV (human herpesvirus 4). When the primary infection occurs in healthy individuals during early childhood, the disease often goes unnoticed. However, when the infection involves healthy adolescent individuals or adults, the resultant disorder is the IM syndrome. Infectious Mononucleosis and Epstein-Barr Virus Infection  Lymphocytopenia  Lymphocytopenia is present when the absolute lymphocyte count is below ≈1.0 × 109/L in adults and below ≈2.0 × 109/L in children. Normally, about 80% of circulating peripheral blood lymphocytes are CD3+ T cells, and a majority (≈65%) of these cells are CD4+ helper T cells. A number of immunologic deficiency disorders that are genetically determined have lymphocytopenia, along with various other immunologic defects of humoral or cell-mediated immunity. Lymphocytopenia in these disorders is due to impaired lymphopoiesis. Increased levels of adrenocortical hormones, administration of chemotherapeutic drugs, or irradiation will result in lymphocytopenia.  In advanced cases of NHL and HL, as well as in terminal cases of carcinoma, lymphocytopenia is often observed. Neoplastic Disorders Primarily Involving Leukocytes Chronic Myelogenous Leukemia  CML occurs in young and middle-aged adults. The age-specific incidence, however, increases markedly after 50 years of age. Onset is insidious, and the disorder may be discovered accidentally on a routine blood test.  The patient may have symptoms of anemia and weight loss or simply may complain of malaise. The spleen enlarges progressively, and the patient begins to lose weight and have fever and night sweats associated with increased metabolism as a result of granulocyte turnover. The discomfort associated with an enlarged spleen may bring the patient to the doctor. Infarcts in the spleen may produce left upper quadrant pain.  Excessive bleeding or bruising may occur in the later stages of the disease. Lymphadenopathy, although often present, is rarely prominent. Chronic Myelogenous Leukemia  The leukocyte count is usually over 5 × 109/L and may exceed 30 × 109/L. The differential count is characteristic. There is a complete spectrum of granulocytic cells, from a few myeloblasts to mature neutrophils, with myelocytes and neutrophils exceeding the other cell types.  This bimodal distribution helps to exclude other myeloproliferative disorders and reactive leukocytosis.  Myeloblasts account for less than 10% of the cells. The relative percentage of neutrophil myelocytes increases as the total leukocyte count increases. Basophilia is consistently present and eosinophilia is almost always noted, along with the presence of eosinophil myelocytes. Monocytes are also absolutely increased in most patients.  Normocytic anemia is present in the majority of patients at diagnosis, and a few normoblasts can usually be found. Thrombocytosis is present in more than half, and less than 15% have thrombocytopenia.  Chronic Myelogenous Leukemia  The marrow is markedly hypercellular, primarily as a result of granulocytic proliferation, with all stages represented.  It is good to remember that even a typical BM is not diagnostic of CML. On the other hand, the diagnosis can be made from the peripheral blood film in most cases. Chronic Myelogenous Leukemia The neutrophil alkaline In more than 95% of patients with typical CML, cultured cells from the phosphatase (NAP) is greatly blood or bone marrow possess the reduced or absent in more than cytogenetic abnormality 90% of patients with CML. It is t(9;22)(q34;q11), involving the ABL1 gene on the long arm of greatly elevated in polycythemia chromosome 9 and the BCR gene vera; elevated, normal, or low in on the long arm of chromosome 22. idiopathic myelofibrosis; and An abnormally small chromosome normal or elevated in leukemoid formed by this translocation is called the Philadelphia (Ph′) reactions. chromosome.  Acute Myeloid Leukemia  AML is the most common form of acute leukemia during the first few months of life, but during childhood and adolescence it accounts for approximately one third of AL. In the middle and later years of life, it becomes the most frequent AL, with a median age of 60, and an occurrence of 10/100,000 per year in those older than 60 years. Viruses, radiation, cytotoxic chemotherapy, benzene, and smoking have been linked to increased incidence, but most cases are not known to be associated with such factors.  Acute Myeloid Leukemia  The onset often resembles acute infection and includes signs of granulocytic insufficiency, with ulcerations of mucous membranes (especially of the mouth and throat) and fever. Enlargement of lymph nodes, spleen, and liver is not pronounced. Marked prostration and general malaise may be present. In untreated cases, the course is rapidly progressive.  The diagnosis of AML requires the presence of 20% blasts in the marrow or blood.  More mature myeloblasts can also be identified by cytochemical reactions with granulocyte-associated enzymes using MPO, Sudan black B (SBB), and chloroacetate esterase (CAE) assays, although flow cytometric assays are utilized more often in current practice. Acute Myeloid Leukemia Acute Myeloid Leukemia Acute lymphoblastic leukemia  Acute lymphoblastic leukemia is the most common malignancy of children and adolescents.  Clinical symptoms - fatigue, fever, and bleeding.  Anemia is present in precursor B-ALL if clinical manifestations are fully developed. It is usually normocytic. Frequently, nucleated red cells are present.  Thrombocytopenia of moderate to marked degree is the rule. The leukocyte count occasionally is very high (>100 × 109/L) and often is slightly elevated, but it is perhaps most frequently normal or decreased. The predominant cell is the lymphoblast.  Acute lymphoblastic leukemia - marrow  By the time the patient is symptomatic, hematopoietic cells and fat are usually replaced by a diffuse infiltration of lymphoblasts. Blast percentage is usually greater than 50%. Predominance of small blasts with high nuclear/cytoplasmic (N/C) ratios and inconspicuous nucleoli (L1 type in the FAB classification) is most common in childhood ALL.  Chromatin is diffuse in some cells but may show variable condensation, and blasts may be difficult to distinguish from the normal lymphocytes of young children. Larger blasts with more abundant cytoplasm, prominent nucleoli, and often irregular nuclei (FAB L2) also occur and tend to predominate in adult ALL.  The blasts are negative for SBB, peroxidase, and naphthol ASD CAE.  Blasts characteristically express TdT, cytoplasmic CD22 and CD79a, CD19, and HLA-DR. CD10 (common ALL antigen) is expressed in many, and expression of CD34 is variable Acute lymphoblastic leukemia Acute lymphoblastic leukemia  The evolution of treatment with combination chemotherapy, CNS treatment, and intensified therapy for high-risk categories has led to cure rates of nearly 80% in children.  Favorable factors are age 5–10 years, hyperdiploidy (best, 54–62 with trisomy 4, 10, and/or 17), t(12;21), and normal or low WBC.  Poor risk factors include age younger than 1 year, t(9;22), and t(4;11).  Among adults, only 30%–40% are cured, in part because of the higher frequency of adverse genetic abnormalitiesл  Adult ALL incidence increases in middle to older age, similarly to genetically high-risk AML  Acute Leukemia  The diagnosis of acute leukemia relies on enumeration of the percentage of blasts in the peripheral blood or bone marrow; the current criterion in the WHO classification for the diagnosis of acute leukemia is greater than 20% blastsл  In flow cytometry, identification of blasts relies on the demonstration of expression of immature antigens by a population having appropriate CD45 expression and light scatter characteristics. Although it is the overall immunophenotype that allows identification of blasts, antigens commonly used for blast identification include CD34, CD117, CD133, and terminal deoxynucleotidyl transferase (TdT)л  Acute Leukemia The major role for flow cytometry in acute leukemia is classification. Classification is largely a matter of lineage assignment and correlation with normal maturational stage. The determination of lineage in particular is a decision of major therapeutic importance, with the primary distinction being whether the leukemia is of myeloid or lymphoid lineage. The immunophenotypic characteristics of acute myeloid leukemia ( Acute Leukemia-flow cytometry Acute Leukemia-flow cytometry  In rare cases, the leukemic cells may show differentiation along more than one lineage, most commonly either myeloid and B cell or myeloid and T cell, as evidence of the stem cell nature of these forms of acute leukemia. If two abnormal blast populations are present, each having a distinct and different lineage, this is termed bilineal acute leukemia. Acute Leukemia-flow cytometry  In rare cases, the leukemic cells may show differentiation along more than one lineage, most commonly either myeloid and B cell or myeloid and T cell, as evidence of the stem cell nature of these forms of acute leukemia. If a single blast population shows such differentiation, it is commonly termed biphenotypic acute leukemia.  Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma  This is a clonal proliferation of small B lymphocytes involving BM, blood, and lymph nodes.  It is rare in those younger than age 40; most cases occur over the age of 60, and the condition is more than twice as common in men as in women. Onset is insidious, and the disease is commonly discovered by chance during the investigation of another problem. Most patients are >50 years of age.  Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma  The leukocyte count is usually between 30 and 20 × 109/L. In the typical type of CLL, 90% or more of the cells are small lymphocytes that are monotonously similar in appearance and usually look normal.  Often there is neither anemia nor thrombocytopenia at the time of diagnosis.  Cytochemically, these cells contain acid phosphatase, which is resistant to inhibition by tartrate-resistant acid phosphatase; this is in contrast to the isoenzymes of acid phosphatase present in other hemic cells  The median survival for patients with CLL is about 7 years, but recent therapy such as fludarabine shows promise for better survival. ЧЕСТИТА БАБА МАРТА!  CLINICAL LABORATORY HEMOSTASIS- PHYSIOLOGIC HEMOSTASIS VERSUS CLINICAL ASSAYS YANA BOCHEVA, MD, PHD DEPARTMENT OF COMMON MEDICINE AND CLINICAL LABORATORY MEDICAL UNIVERSITY-VARNA  HEMOSTASIS Hemostasis, or cessation of bleeding, occurs within the intravascular compartment lined with endothelium. Normal hemostasis and thrombosis involve a number of factors. These factors include platelets, granulocytes, and monocytes, as well as the coagulation (clot forming), fibrinolytic (clot lysing), and anticoagulant (regulating) protein systems. The coagulation system serves to form thrombin that initiates the proteolysis of fibrinogen, leading to fibrin clot formation. The role of the fibrinolytic system is to lyse the clot formed by thrombin. The role of the anticoagulation system is to regulate all enzymes of the coagulation and fibrinolytic systems, so that no inappropriate excess of clotting or bleeding occurs. These three protein systems, together with the vessel wall endothelium, are in delicate balance to ensure adequate hemostasis with limitation on thrombosis formation. HEMOSTASIS When a vessel is severed or punctured, or when the wall of a vessel is damaged, vascular spasm occurs. It is triggered by several chemicals called endothelins that are released by vessel-lining cells and by pain receptors in response to vessel injury. It lasts for up to 30 minutes. HEMOSTASIS In the second step the platelets begin to clump together, become spiked and sticky, and bind to the exposed collagen and endothelial lining. This process is assisted by a glycoprotein in the blood plasma called von Willebrand factor, which helps stabilize the growing platelet plug. A platelet plug can temporarily seal a small opening in a blood vessel. HEMOSTASIS Those more sophisticated and more durable repairs are collectively called coagulation, the formation of a blood clot. The process is sometimes characterized as a cascade, because one event prompts the next as in a multi-level waterfall. The result is the production of a gelatinous but robust clot made up of a mesh of fibrin—an insoluble filamentous protein derived from fibrinogen, the plasma protein introduced earlier— in which platelets and blood cells are trapped.  HEMOSTASIS In the coagulation cascade, chemicals called clotting factors (or coagulation factors) prompt reactions that activate still more coagulation factors. The process is complex, but is initiated along two basic pathways: The extrinsic pathway, which normally is triggered by trauma. The intrinsic pathway, which begins in the bloodstream and is triggered by internal damage to the wall of the vessel Both of these merge into a third pathway, referred to as the common pathway. All three pathways are dependent upon the 12 known clotting factors, including Ca2+ and vitamin K Clotting factors are secreted primarily by the liver and the platelets. The liver requires the fat-soluble vitamin K to produce many of them CLOTTING FACTORS  EXTRINSIC PATHWAY The quicker responding and more direct extrinsic pathway (also known as the tissue factor pathway) begins when damage occurs to the surrounding tissues, such as in a traumatic injury. Upon contact with blood plasma, the damaged extravascular cells, which are extrinsic to the bloodstream, release factor III (thromboplastin). Sequentially, Ca2+ then factor VII (proconvertin), which is activated by factor III, are added, forming an enzyme complex. This enzyme complex leads to activation of factor X (Stuart–Prower factor), which activates the common pathway discussed below. The events in the extrinsic pathway are completed in a matter of seconds.  INTRINSIC PATHWAY The pathway can be prompted by damage to the tissues, resulting from internal factors such as arterial disease; however, it is most often initiated when factor XII (Hageman factor) comes into contact with foreign materials, such as when a blood sample is put into a glass test tube. Within the body, factor XII is typically activated when it encounters negatively charged molecules, such as inorganic polymers and phosphate produced earlier in the series of intrinsic pathway reactions. Factor XII sets off a series of reactions that in turn activates factor XI (antihemolytic factor C or plasma thromboplastin antecedent) then factor IX (antihemolytic factor B or plasma thromboplasmin). In the meantime, chemicals released by the platelets increase the rate of these activation reactions. Finally, factor VIII (antihemolytic factor A) from the platelets and endothelial cells combines with factor IX (antihemolytic factor B or plasma thromboplasmin) to form an enzyme complex that activates factor X (Stuart–Prower factor or thrombokinase), leading to the common pathway. The events in the intrinsic pathway are completed in a few minutes.  COMMON PATHWAY Both the intrinsic and extrinsic pathways lead to the common pathway, in which fibrin is produced to seal off the vessel. Once factor X has been activated by either the intrinsic or extrinsic pathway, the enzyme prothrombinase converts factor II, the inactive enzyme prothrombin, into the active enzyme thrombin. (Note that if the enzyme thrombin were not normally in an inactive form, clots would form spontaneously, a condition not consistent with life.) Then, thrombin converts factor I, the insoluble fibrinogen, into the soluble fibrin protein strands. Factor XIII then stabilizes the fibrin clot  FIBRINOLYSIS The stabilized clot is acted upon by contractile proteins within the platelets. As these proteins contract, they pull on the fibrin threads, bringing the edges of the clot more tightly together, somewhat as we do when tightening loose shoelaces. This process also wrings out of the clot a small amount of fluid called serum, which is blood plasma without its clotting factors. To restore normal blood flow as the vessel heals, the clot must eventually be removed. Fibrinolysis is the gradual degradation of the clot. Again, there is a fairly complicated series of reactions that involves factor XII and protein-catabolizing enzymes. During this process, the inactive protein plasminogen is converted into the active plasmin, which gradually breaks down the fibrin of the clot. Additionally, bradykinin, a vasodilator, is released, reversing the effects of the serotonin and prostaglandins from the platelets. This allows the smooth muscle in the walls of the vessels to relax and helps to restore the circulation  PLASMA ANTICOAGULANTS An anticoagulant is any substance that opposes coagulation. Several circulating plasma anticoagulants play a role in limiting the coagulation process to the region of injury and restoring a normal, clot-free condition of blood. For instance, a cluster of proteins collectively referred to as the protein C system inactivates clotting factors involved in the intrinsic pathway. TFPI (tissue factor pathway inhibitor) inhibits the conversion of the inactive factor VII to the active form in the extrinsic pathway. Antithrombin inactivates factor X and opposes the conversion of prothrombin (factor II) to thrombin in the common pathway. And as noted earlier, basophils release heparin, a short-acting anticoagulant that also opposes prothrombin. Heparin is also found on the surfaces of cells lining the blood vessels. A pharmaceutical form of heparin is often administered therapeutically, for example, in surgical patients at risk for blood clots. CLINICAL LABORATORY HEMOSTASIS When faced with a bleeding patient, one must use an analytic diagnostic approach to determine the cause of the problem. The underlying cause in almost all cases will derive from a defect or deficiency in a plasma protein, a defect in platelet number or function, or a defect in adhesive interactions between platelets and the vessel wall. Any coagulation protein defect can be a true protein deficiency, an inhibitor to the active site of the protein, an abnormal protein that cannot participate in its physiologic function(s), or an apparent deficiency that arises as the result of enhanced clearance of protein. In general, inhibitors to a coagulation protein are immunoglobulins, although hypergammaglobulinemic states or abnormal production of endogenous heparin, fibronectin, or cryoglobulins as acquired inhibitors to coagulation proteins have also been reported. Abnormal proteins that are present but do not function normally occur as a result of missense or deletion mutations, or translocations of DNA. Last, enhanced clearance of coagulation proteins usually occurs as a result of antigen–antibody complex formation. Resultant increased clearance of the protein gives the appearance of a deficiency, but the real mechanism is enhanced clearance PATTERNS OF CLINICAL BLEEDING IN DISORDERS OF HEMOSTASIS In general, hemarthrosis and spontaneous soft tissue and intramuscular hemorrhage characterize plasma protein defects such as hemophilia A and B (factors VIII and IX deficiency). Soft tissue petechiae, purpura, or ecchymosis characterizes von Willebrand disease, or disorders of platelet number or function. However, at times, it is difficult to distinguish the potential mechanism for bleeding. Thus the clinical laboratory is essential for definitive diagnosis of a bleeding disorder in the patient.  SCREENING TESTS FOR COAGULATION DISORDERS The term primary hemostasis refers to platelet reactivity at the site of vessel injury. The term secondary hemostasis is connected with the coagulation of plasma due to coagulation factors. In this hypothesis, coagulation proteins are classified as members of the so-called intrinsic system, extrinsic system, or common pathway  SCREENING TESTS FOR COAGULATION DISORDERS  ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) To perform this common coagulation assay, a mixture of a negatively charged surface, phospholipid, and anticoagulated patient plasma is incubated for several minutes. The recommended anticoagulant is 3.2 g% sodium citrate because less variation is seen in blood specimens from normal patients and those on anticoagulants collected in this concentration of anticoagulant. Sodium citrate is a reversible chelator of calcium that prevents coagulation protein activation. When whole blood is collected, the ratio of anticoagulant to whole blood is 1 part anticoagulant to 9 parts whole blood. After incubation of patient plasma with reagent for a prescribed time, depending on the assay, the sample is recalcified with excess calcium chloride, and the time required for clot formation is measured. The APTT assesses the coagulation proteins of the so-called intrinsic system and common pathways (see later). This assay is commonly referred to as the partial thromboplastin time (PTT), but it is really an “activated” PTT, in that its reagents contain a negatively charged surface that accelerates the rate of the reaction.  PROTHROMBIN TIME To perform this common coagulation assay, tissue thromboplastin (recombinant human or isolated animal tissue factor) and patient plasma are incubated for several minutes, after which the citrated plasma mixture is recalcified by the addition of excess CaCl2, and the time required for clot formation is measured. The PT assesses the coagulation proteins of the so-called extrinsic system and common pathway. Tissue thromboplastin traditionally has been a crude preparation of animal brain TF. Presently, recombinant TF is used in the preparation of several commercial PT reagents. In general, the range of prolongation of time of an abnormal PT increases when recombinant TF is used. This fact makes for a more sensitive assay. The PT serves as the basis for the international normalized ratio (INR) value used to monitor patients on warfarin. INR is the ratio of patient PT divided by geometric mean normal PT for the local laboratory (based on a population of normal individuals assessed with identical sample collection, reagents, and machines), raised to the power of the international sensitivity index. Although the INR is clearly the most appropriate measure to use in conjunction with oral anticoagulant monitoring, for hemostatic evaluation of the non-warfarinized patient, actual PT values in seconds may be used, referencing the laboratory’s locally established reference interval for the PT test INR CALCULATION  THROMBIN TIME To perform the thrombin time (also referred to as thrombin clotting time), purified exogenous thrombin is added to plasma to determine the time to clot formation. It is a direct measure of fibrinogen function and may be used to ascertain if there is a defect in fibrinogen function. The thrombin time will be prolonged in hypo-fibrinogenemic states, if an abnormal protein fibrinogen (dysfibrinogenemia) is present, or if a thrombin inhibitor is present.  CLINICAL COAGULATION TESTING Clinical coagulation testing is based on functional assays that examine the rate of clot formation. In these assays, a sequence of proteolytic reactions takes place, leading to thrombin formation and its proteolysis of fibrinogen. All coagulation factors of the intrinsic system (XII, prekallikrein, high molecular weight kininogen, XI, IX, and VIII) are measured in assays using the APTT as its platform. For example, a FVIII assay is a mixture of APTT reagent, FVIII-deficient plasma, and test (unknown) plasma from the patient.  CLINICAL COAGULATION TESTING FVII, together with coagulation factors of the common pathway (X, V, and II), is usually measured by assays using the PT as the platform. For example, an FX assay is a mixture of PT reagent that contains TF (thromboplastin), FX-deficient plasma, and test or patient (unknown) plasma. Coagulation-based assays are sensitive and specific. They are much simpler to perform than antigen assays for each of the coagulation proteins. These assays provide information on the functional presence of the coagulation protein.  CLINICAL COAGULATION TESTING Coagulation-based assays examine the function of the protein; antigen assays establish the presence of the proteins; combined, the two assays characterize any protein with reduced function but with normal antigen. A dysfunctional protein has reduced protein function with normal levels of antigen. Such a situation arises commonly when fibrinogen is examined. The most useful means to determine whether fibrinogen is abnormal (i.e., dysfibrinogenemia) is to measure clottable fibrinogen and fibrinogen antigen. If fibrinogen clottability is less than 90% of the amount of fibrinogen antigen present, this finding would suggest that the protein produced is abnormal in some way.  CLINICAL COAGULATION TESTING Chromogenic assays are used to measure certain enzymes (plasmin, activated protein C) and various plasma protease inhibitors (antithrombin, C1 inhibitor, α2-antiplasmin, PAI, tPA). Presently, such assays are used to measure the therapeutic levels of unfractionated heparin, low molecular weight heparin, or fondaparinux. DIFFERENTIAL DIAGNOSIS OF ABNORMAL COAGULATION SCREENING TESTS  PT The PT measures the extrinsic coagulation pathway of coagulation, which consists of activated FVII (FVIIa) and TF and proteins of the common pathway (factors X, V, II, and fibrinogen). FVII levels below 35%–40% will begin to be detected by prolongation of the PT. The thrombin time measures only the ability of exogenous thrombin to proteolyze (clot) fibrinogen. It is used to characterize fibrinogen function  APTT If a patient has an isolated prolonged APTT, determination of the patient’s risk to bleed can begin with the addition of some historical information. If isolated prolongation of the APTT in a male patient is associated with bleeding, then the differential diagnosis in decreasing likelihood of frequency is FVIII, FIX, or FXI deficiency. Von Willebrand disease is the most common bleeding disorder in humans, that can cause prolongation of APTT. If the APTT alone is prolonged and there is no history of bleeding, the most common cause is a lupus anticoagulant. The specific proteins of the so-called intrinsic coagulation system associated with a prolonged APTT but no bleeding history include, in decreasing frequency, FXII, prekallikrein, and high molecular weight kininogen. Knowing about these latter three proteins is essential in evaluating a prolonged APTT even though the patient is not at bleeding risk, so that patients neither get unnecessary plasma replacement therapy nor experience unnecessary delays in scheduled surgical procedures.  PT AND APTT Defects in some common pathway proteins (fibrinogen, factors II, V, and X) may first produce an isolated PT prolongation, although, if severe, these latter protein defects will lead to prolongations of the PT and APTT. When confronted with patient laboratory results of prolonged PT and APTT, it is important not simply to consider the specific proteins mentioned earlier but also to address the differential diagnosis from general medical states such as anticoagulation therapy, disseminated intravascular coagulation, liver disease, vitamin K deficiency, and massive transfusion.  HEREDITARY COAGULATION PROTEIN DEFECTS Protein or factor deficiencies can be quantitative or qualitative. In quantitative disorders, the factor level determined by routine clot-based methods (functional activity assays) is similar to that obtained by immunologic (antigen) assays. In qualitative disorders, the functional assay result is decreased, but the antigen level is significantly higher or normal, indicating the presence of a dysfunctional protein or an inhibitor to the function of that protein.  VON WILLEBRAND DISEASE Von Willebrand disease (VWD) is the most common hereditary blood-clotting disorder in humans.  LABORATORY TEST IN VON WILLEBRAND DISEASE complete blood count-CBC (especially platelet counts), activated partial thromboplastin time-APTT, prothrombin time with International Normalized Ratio-PTINR, thrombin time-TT, and fibrinogen level. When VWD is suspected, blood plasma of a patient must be investigated for quantitative and qualitative deficiencies of VWF. This is achieved by measuring the amount of VWF in a VWF antigen assay and the functionality of VWF with a glycoprotein (GP)Ib binding assay, a collagen binding assay, or a ristocetin cofactor activity (RiCof) or ristocetin-induced platelet agglutination (RIPA) assays. Factor VIII levels are also performed because factor VIII is bound to VWF which protects the factor VIII from rapid breakdown within the blood. Deficiency of VWF can then lead to a reduction in factor VIII levels, which explains the elevation in PTT. ACQUIRED VON WILLEBRAND DISEASE Most patients with acquired vWD (AvWD) have been older than 40 years of age without previous manifestations or family history of a bleeding diathesis. Diverse associated disorders in these patients include both benign and malignant hematologic disorders (Federici, 2000; Kumar, 2002). About half of patients have had an underlying lymphoproliferative disorder or plasma cell proliferative disorder (Federici, 2000). In patients with myeloproliferative disease (chronic myelogenous leukemia, essential thrombocythemia, polycythemia vera) and with reactive thrombocytosis, an impressive correlation has been noted between abnormalities in plasma vWF and elevated platelet counts (Budde, 1997). AvWD has been reported in patients with autoimmune disorders, including systemic lupus erythematosus, scleroderma, mixed connective tissue disease, hypothyroidism, and antiphospholipid antibody syndrome  HEMOPHILIA „Kitab al-Tasrif“Абу Бакр Мохамед ибн Закария ал-Рази – Албуказис. QUEEN VICTORIA Victoria was the daughter of Prince Edward, Duke of Kent and Strathearn, the fourth son of King George III. She inherited the throne at the age of 18, after her father's three elder brothers had all died, leaving no surviving legitimate children. Victoria married her first cousin Prince Albert of Saxe- Coburg and Gotha in 1840. Their nine children married into royal and noble families across the continent, tying them together and earning her the sobriquet "the grandmother of Europe". Her reign of 63 years and seven months was longer than that of any of her predecessors and is known as the Victorian era.  HEMOPHILIA A /HEMOPHILIA B Bleeding manifestations of hemophilia A and B, which are coagulation factor problems, include hemarthrosis; soft tissue hematomas into muscles; easy bruising; excessive bleeding with surgery, trauma, dental extraction, and circumcision; bleeding in the gastrointestinal or genitourinary tract; epistaxis; poor wound healing; and, uncommonly, umbilical stump bleeding. Intracranial hemorrhage can occur, particularly following trauma. The severity of hemophilia is classified on the basis of plasma factor level. Severe hemophilia presents with spontaneous bleeding two to four times per month and requires frequent treatment or prophylactic therapy with replacement factor products. At the other end of the spectrum, mild hemophilia presents with prolonged bleeding after trauma or major surgery, and patients rarely need intravenous factor replacement. CLASSIFICATION OF HEMOPHILIA A AND B  HEMOPHILIA Hemophilia is suspected on the basis of bleeding symptoms or a family history of hemophilia. About one third of hemophilia A cases arise from spontaneous mutations. Laboratory evaluation of patients with such a bleeding history should include an APTT and PT. Diagnosis is confirmed by a FVIII or FIX assay. More diagnostic testing may be required for patients with mild to moderate deficiency of FVIII for whom a diagnosis of hemophilia A may be less apparent (e.g., a female patient with low FVIII and apparent autosomal inheritance). In patients with von Willebrand disease, a secondary deficiency of FVIII may occur , because FVIII is normally bound to von Willebrand factor in the plasma  HEMOPHILIA FIX activity levels during childhood remain at about 75% of adult levels. A 25% increment in FIX expression begins at puberty in both sexes. It has been assumed that this is a result of steroid hormone action. It is interesting to note that a rare form of FIX deficiency, hemophilia B Leyden, undergoes postpubertal phenotypic resolution. Patients with this condition present with hemophilia B in early childhood, with FIX activity ranging from less than 1%–13% of normal. Plasma levels rise to as high as 70% of normal after the onset of puberty with resolution of bleeding complications.  TREATMENT/COMPLICATIONS OF TREATMENT OF HEMOPHILIA Recombinant factor products are now the standard of care for treatment of hemophilia A and B in developed countries. Inhibitor antibodies to FVIII or FIX represent significant complications of therapy. The incidence of inhibitors in patients with severe hemophilia A is approximately 15%–35%, and the incidence of inhibitors in patients with hemophilia B is approximately 1%–4%.  DISORDERS OF FIBRINOGEN Fibrinogen deficiencies may prolong the PT and APTT if the plasma concentration of the protein is sufficiently low, usually less than 100 mg/dL. Afibrinogenemia is autosomal recessive and represents the total absence of fibrinogen. It results in a bleeding disorder of variable severity. Umbilical stump and mucosal bleeding are most common, as is an increased incidence of musculoskeletal and central nervous system bleeding. Patients also exhibit poor wound healing. Hypofibrinogenemia is a decreased level of normal fibrinogen and has a similar but milder pattern of bleeding. Bo