1 Dr Amani Abdelfattah M.D in Clinical Pathology Causes of misleading results in hematology lab

2 Hematology lab tests Frequently done tests CBC, CBC with differential Peripheral smear Reticulocyte count Prothrombin time Partial thromboplastin time Fibrinogen assay Erythrocyte sedimentation rate Malaria screen Hemoglobin electrophoresis Second line investigations Correction tests (mixing studies). Lupus anticoagulant assay. Coagulation factors assays Antithrombin III, Protein C, Protein S assays and APCR assays. Heparin induced thrombocytopenia. Haemoglobin S solubility test. Bone marrow Examination. Immunophenotyping.

3 Hematology lab tests Frequently done tests CBC

4 Hematology lab tests Frequently done tests Reticulocyte count Reticulocytes are juvenile red cells; they contain remnants of the ribosomal ribonucleic acid (rRNA) that was present in larger amounts in the cytoplasm of the nucleated precursors from which they were derived.

5 Hematology lab tests Frequently done tests Prothrombin time It measures the clotting time of recalcified plasma in the presence of an optimal concentration of tissue extract (thromboplastin) and indicates the overall efficiency of the extrinsic clotting system.

6 Hematology lab tests Frequently done tests Partial thromboplastin time The test measures the clotting time of plasma after the activation of contact factors and the addition of phospholipid and CaCl2 but without added tissue thromboplastin and so indicates the overall efficiency of the intrinsic pathway.

7 Hematology lab tests Frequently done tests ESR Inflammation, infections Pregnancy, Anemia autoimmune disorders Some cancers (such as lymphoma and multiple myeloma). Polycythemia Sickle cell anemia

8 Hematology lab tests Frequently done tests Malaria screen Plasmodium falciparum ring and gametocyte thin blood smear Plasmodium vivax schizont in thin blood smear

9 Hematology lab tests Frequently done tests Hemoglobin electrophoresis For investigationof ubnormal hemoglobins and thalassaemia High performance liquid chromatography

10 Hematology lab tests Second line investigations Mixing studies (correction test) are tests performed on blood plasma when there is unexplained prolongation of PT or aPTT by adding equal volum of patient plasma and normal pooled plasma Used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as fVIII inhibitor

11 Hematology lab tests Second line investigations Coagulation factors assays FVIII,FIX,vWF,FV FX,FII,FXI,FXII,FXIII Investigation of Thrombotic Tendency lupus anticoagulant Antithrombin,Protein C and Protein S assays Detection of markers of coagulation activation D-dimer

12 Hematology lab tests Second line investigations Haemoglobin S solubility test Sickle cell haemoglobin is insoluble in the deoxygenated state in a high molarity phosphate buffer. The crystals that form refract light and cause the solution to be turbid.

13 Hematology lab tests Second line investigations Heparin induced thrombocytopenia also known heparin PF4 antibody Day 1Day 4Day 14 Rapid-onset HIT (hours–days) Typical HIT Mean Day 9 (4–14 days) Heparin (re) Exposure THROMBOCYTOPENIA (± THROMBOSIS )

14 Hematology lab tests Second line investigations Bone marrow examination

15 Hematology lab tests Second line investigations Imunophenotyping It is a technique used to study the protein expressed by cells An example is the detection of tumor marker, such as in the diagnosis of leukemias.

16 Laboratory test results Good Laboratory Practice Clinical diagnosis Patient management

17 Causes of misleading results in hematology lab

18 Pre-analytical phase errors Sample collection Patient Identification problems Sampling technique Sample condition (diluted- insufficient-overfilled- hemolysed-clotted, etc.,) Blood collection tubes Order of drawing Sample mixing Sample handling and transport Sample centrifugation Sample storage Variables related to the patient Pre collection stress and exercise pregnancy Smoking Circadian variation

19 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Patient Identification problems Mislabeling is a serious error collection from the wrong patient specimen mix-up transcription error These can occur at any stage. It is essential to have a cross-check procedure.

20 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Time of tournique application Applying the tourniquet >1minute cause hemoconcentration and false increase in vWF and F VIII Patient position during sampling There are difference in CBC (Hb,Hct and WBCs ) between lying down and sitting position

21 Drawing blood samples from an arm with running transfusion : If an arm with a running transfusion must be used Wait 1 hour after transfusion is complete to draw specimen When an intravenous solution is being administered in a patient's arm, blood should be drawn from the opposite arm. If an intravenous infusion is running in both arms, samples may be drawn after the intravenous infusion is turned off for at least 2minutes before venipuncture and applying the tourniquet below the intravenous infusion site. Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Sample condition

22 Drawing samples from a central line : If there is no alternative way first flush it with saline then discard the initial sample to avoid sample dilution and heparin contamination if the line contains heparin Drawing blood samples for coagulation study by butterfly” Drawing a discard tube first is a must

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24 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Blood collection tubes Sample for coagulation studies must be collected only in the blue top tube as it contains 3.2% Sodium Citrate which is the anticoagulant of choice for coagulation assays It is critical to stick to anticoagulant/blood ratio (1:9) No under filled or over filled are acceptable NEVER combine two underfilled tubes to make one filled tube Sampling must be done according to the recommended order of draw

25 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Blood collection tubes K 2 EDTA

26 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Sample mixing

27 Causes of misleading results in hematology lab Pre-analytical phase errors ( 46-68.2%) Sample handling and transport Samples should be sent within 1hour of collection at room temperature. Sending samples on ice will activate the sample = shortened clotting times Exposure of samples to excess heat e.g. storage next to a radiator or exposure to strong sunlight through a car, can cause denaturation of plasma proteins

28 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Sample receiving Laboratory checks for quantity, quality, labeling, request forms, clot presence etc If these criteria are not met then the samples may be rejected

29 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Sample rejection policy Labeling Problem Unacceptable Container Unacceptable Specimen: Inappropriate volume of blood (Over filled) or under filled tubes. Hemolysis samples Clotted sample Unacceptable transportation time, temperature Normally Sterile Body Fluids (i.e.: pericardial, peritoneal, CSF) Bone Marrow Bronchoscopy/Endoscopy Specimens (i.e., BAL's Will be processed under a temporary provision which requires the collector to come to the laboratory and fill out the appropriate form acknowledging responsibility and verifying the identity of the specimen. This procedure must be completed before the results will be posted and should be documented Specimens which can be re-obtained Specimens which cannot be re-obtained

30 Causes of misleading results in hematology lab Pre-analytical phase errors (46-68.2%) Sample centrifugation Proper centrifugation Specimen must be centrifuged for at least 15 minutes @ 4000 RPM to obtain Platelet Poor plasma Centrifugation must done within one hr of collection Why is PPP essential? Because Platelets Contains Platelet factor 4 (heparin neutralizer) Phospholipid (affects lupus anticoagulant assay)

31 Causes of misleading results in hematology lab Pre-analytical phase errors Effect of storage on coagulation assays Coagulation test stability is critical for diagnosis and treatment of coagulopathies; it is recommended that Tests be carried out within 2 h when the blood or plasma is stored at 22–24 °C. within 4 h when stored at 4 °C. Within 2 weeks when stored at −20 °C within 6 months when stored at −70 °C.3 Repeated freeze-thaw cycles may decrease vWF activity and FXII levels.

32 Causes of misleading results in hematology lab Pre-analytical phase errors Effect of storage on CBC parameter Parameter HGB RBC count MCV HCT PLT Reticulocytes (RET) WBC Storage at 4 °C Stable up to 72 h Stable up to 72 h Stable up to 6 – 12 h Stable up to 24 h Stable up to 72 h Stable 24 up to 72 h It is important to make films as soon as possible, with 1 hour

33 Causes of misleading results in hematology lab Pre-analytical phase errors Effect of storage on RBCs morphology Normal RBCs are morphologically stable for up 6 h at room temperature longer period leads to crenation, sphering (EDTA salts mainly affects the size of RBCs due to an osmotically dependent shrinkage). It is reported that nucleated red blood cells (NRBCs) disappear from the blood specimen within 1 or 2 days. All of the above changes are retarded but not abolished in samples stored at 4 °C. http://www.medical-labs.net/wp- content/uploads/2014/01/Crenated-Cells.jpg

34 Causes of misleading results in hematology lab Pre-analytical phase errors Effect of storage on WBCs morphology By 3 h, changes may be noticed - Changes in neutrophils :separation of the nuclear lobes,the cytoplasmic margin appear ragged or less well- defined, small vacuoles appear in the cytoplasm -Changes in monocytes and lymphocytes : small vacuoles appear in the cytoplasm and the nucleus undergoes irregular lobulation

35 Causes of misleading results in hematology lab Pre-analytical phase errors Variables related to the patient Stress and exercise FVIII &t-PA WBCs and platelets aggregation to ADP and epinephrine

36 Causes of misleading results in hematology lab Pre-analytical phase errors Variables related to the patient Circadian variation FVIII activity, FVIIa PC,PS Fibrinolysis the early morning

37 Causes of misleading results in hematology lab Pre-analytical phase errors Variables related to the patient Effect of pregnancy FVII,FVIII,Vwf, Fx Fibrinogen and D- dimer Free protein S

38 Causes of misleading results in hematology lab Pre-analytical phase errors Variables related to the patient Smoking WBC, Hg, RBCs,Hct, MCV,MCH, Platelets count, Fibrinogen ESR Protein S and plasma volume Stop smoking 2 h prior to the venipuncture

39 Causes of misleading results in hematology lab Analytical phase errors (7-13%) With the recent advancement in technology and introduction of automation in hematology laboratories the analytical part of sample analysis is well taken care of if good quality control practices are followed(Maintenance of the analyzer internal quality control (IQC) and external quality control proficiency testing)

40 Causes of misleading results in hematology lab Post-analytical phase errors Post analytical errors like typographical errors can be avoided with laboratory interface systems and hospital information system Post analytical phase also include : Alert the clinician for report with critical values Turn Around Time (TAT) Sample retention Sample disposal Record retention

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42 Thank You

43 43 Wall of the Arteries and veins are composed of three layers Tunica externa: the outer layer composed of connective tissue. Tunica media: the middle layer composed of smooth muscle and elastic tissue. Tunica intima : the inner layer composed of a lining of epithelial cells. The space within a blood vessel through which the blood flows is called a lumen. Most veins are equipped with valve so prevent blood flowing in the reverse direction

44 VeinsArteries Direction of Blood Veins carry deoxygenated blood From various parts of the body to the heart. Arteries carry oxygenated blood From the heart to various parts of the body Anatomy Thin, elastic muscle layer with semilunar valves that prevent the blood from flowing in the opposite direction. Thick, elastic muscle layer that can handle high pressure of the blood flowing through the arteries. LocationCloser to the skinDeeper in the body WallsVeins have collapsible walls Arterial walls are more rigid Dilate and constrict, which creates a pulse Valves Valves present, especially in limbs No valves Store about 65% to 70% of the body’s total blood volume Store about 20% ColorBlood appears darker in color Appear bright red due to high oxygen levels

45 Why routine blood collection performed on veins rather than arteries? Veins are: Generally located closer to the surface in the extremities and are easier to locate Easier to puncture than arteries because vein layers contain less tissue Supplied with fewer nerve fibres than arteries; therefore venipuncture is less painful. Artries are At risk of extended bleeding after puncture because blood flowing through is under great pressure.

46 Superficial veins of the arm Venous access (either for blood collection or intravenous lines) is generally performed on the superficial veins of the arm. The superficial veins of the arm consist of –the digital and metacarpal veins on the back of the hand –Cephalic, basilic and median veins (median cephalic, median basilic, median cubital and median antebrachial). In the forearm and cubital fossa

47 VEINS OF UPPER LIMB SUPERFICIAL VEINS -DORSAL VENOUS NETWORK -CEPHALIC VEIN -BASILIC VEIN -AXILLARY VEIN( BASILIC+ BRACHIAL VEIN) -MEDIAN CUBITAL VEIN -MEDIAN VEIN OF FOREARM -DEEP VEINS -NAMED ACCORDING TO COMPANIAN ARTERIES -RADIAL VEIN -ULNAR VEIN -BRACHIAL VEIN -AXILLARY VEIN

48 Antecubital Fossa shallow depression in the arm that is anterior to the elbow. It is the first-choice location for venipuncture because several major arm veins lie close to the surface, making them relatively easy to locate and penetrate

49 Cephalic Basilic Median cubital Radial nerve Median nerve Brachial artery

50 Antecubital Fossa The anatomical arrangement of antecubital veins varies slightly from person to person; however, two basic vein arrangements, referred to as the H- and M-shaped patterns

51 51 median cubital vein The H-shaped pattern is displayed by approximately 70% of the population involves the cephalic vein on the lateral side of the arm connected to the basilic vein on the medial side by the median cubital vein just below the elbow crease Located near the center of the antecubital area, it is the preferred vein for venipuncture in the H-shaped pattern. It is typically larger, closer to the surface, better anchored, and more stationary than the others, Least painful to puncture and the least likely to bruise.

52 52 Cephalic vein Located in the lateral aspect of the antecubital area, it is the second choice vein for venipuncture in the H-shaped pattern. It is often harder to palpate than the median cubital but is fairly well anchored and often the only vein that can be palpated (felt) in obese patients. Accessory cephalic vein is lateral branch of The cephalic vein. it originates from dorsal venous network on the back of the hand

53 53 Basilic vein Basilic vein: A large vein located on the medial aspect of the antecubital area, it is the last-choice vein for venipuncture. It is generally easy to palpate but is not as well anchored and rolls more easily, increasing the possibility of accidental puncture of medial cutaneous nerve (a major nerve of the arm) or the brachial artery. Punctures in this area also tend to be more painful.

54 54 Represents about 20-30% of individuals The median antebrachial vein drains the venous plexus on the palmar surface of the hands The median antebrachial vein passes up to the centre of the forearm and gives 2 branches 1.the median cephalic :connecting to the cephalic vein, 2.the median basilic: connecting to the basilic vein

55 55 Median antebrachial vein Median vein (antebrachial vein): The first choice for venipuncture in the M- shaped pattern because it is well anchored, tends to be less painful to puncture, Not close to major nerves or arteries as the others, making it generally the safest one to use.

56 56 Median cephalic vein The second choice for venipuncture in the M- shaped pattern it is accessible and located away from major nerves or arteries, making it generally safe to puncture.

57 57 Median basilic vein The last choice for venipuncture in the M- shaped pattern (even though it may appear more accessible) Because it is more painful to puncture and, like the basilic vein, is located near the branches of the medial cutaneous nerve and the brachial artery.

58  The dorsal digital veins drain into dorsal metacarpal veins, which unite to form a dorsal venous arch or network.  Dorsal venous network lies on the dorsum of the hand, in the subcutanous tissue, proximal to the metacarpophalangeal joints  Drains into the cephalic vein laterally, and basilic vein medially Dorsal Venous Arch (network)

59 Dorsal venous arch Metacarpal plexus Hand Veins When the antecubital veins are not accessible. Hand veins are smaller and less anchored. more likely to roll and collapse painful due to increased nerve endings risk of phlebitis Forarm and Hand Veins

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61 61 Because of the potential for significant medical complications such as phlebitis or thrombosis, veins of the leg, ankle, and foot) must not be used for venipuncture without permission from the patient’s physician. Puncture of the femoral vein is performed only by physicians or specially trained personnel.

62 62 Selecting the Venipuncture Site Everybody will not have veins in the exactly same position. Some variations may exist. Always examine the antecubital area first Always examine the antecubital area first Select a vein that is large and does not roll Select a vein that is large and does not roll

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64 Chapter 264 The most commonly used veins for venipuncture are located in the antecubital fossa. 1st Choice Median cubital vein or median antebrachial veins 2nd Choice Cephalic vein 3rd Choice Basilic vein

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66 Soft Straight Refills when depressed Visible and Easily palpable Has a large lumen Well supported (not rolling) Away from artries and nerves Hard Thrombosed, sclerosed, fibrosed,Inflamed, bruised Thin or Fragile Mobile or tortuous Near bony prominences, painful Areas or sites of Hematoma infection, oedema In the lower extremities good vein :A void veins which are:

67 Artery nerve Feet ankle IV infusion Edema Mastectomy Hematoma Scars Fistula shunt Do not go there The risk is too great Areas to be avoided

68 68 Occluded (Blocked) or Sclerosed Veins. Due to: Inflammation, chemotherapy, arteriosclerotic repeated punctures. These veins may be susceptible to infection or produce erroneous test results because of impaired blood flow. Burns and Scars. susceptible to infection painful to the patient difficult to penetrate and locate inaccurate test results Areas to be avoided

69 69 Edematous Tissue. Take from other arm Fistulas. Renal dialysis patients Must avoid this arm Possibility of infection Areas to be avoided patient with Intravenous Therapy. stop the infusion Wait at least 15 min blood should be drawn below the IV with the tourniquet also placed below the IV site

70 70 Mastectomy patient Risk of Lymphostasis, and lymphedema susceptible to infection Never use a tourniquet The CLSI requires physician consultation before venipuncture Areas to be avoided

71 Remember : Avoid any areas of disruption in the skin such as open lesions, rashes, recent tattoos, or incompletely healed stitches.

72 72 Nerve injury Patient may feel sharp electric pain at venipuncture site Stop the procedure immediately Apply firm pressure, for at least 15minutes After bleeding has stopped, an ice pack may be applied to decrease inflammation. Fill out an incident report Patient may need physical therapy. The phlebotomist must select the appropriate veins for venipuncture and should not blindly probe the arm. Areas to be avoided

73 73 Accidental Arterial puncture Suspected when Fast blood-flow rate Bright red blood. pulsating needle development of a hematoma Areas to be avoided May result in late complications such as arteriovenous fistulae or pseudoaneurysm or finger necrosis

74 74 Accidental Arterial puncture Stop the process and release tourniquet immediately, Apply firm pressure, for at least 5minutes, Fill out an incident report Areas to be avoided

75 75 Difficulty in Finding a Vein Always check both arms. Massage the arm from the wrist to the elbow. Dangle the arm in a downward position. Rotate the wrist to check the cephalic vein. Apply heat Use a blood pressure cuff. (do not inflate to more than 80 mm Hg) ask the patient where the best place to collect blood is located. Try Vein Illumination device if available Physician permission is required for foot or leg punctures

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