Hematology Tips (1): Introduction, CBC (FBC), Hemolysis

Dr. James Manos (MD)
January 2, 2016

(corrected: 26 August 2018)

        Tips in Hematology
          Volume (1)

2^nd edition (revised)

CONTENTS

INTRODUCTION IN HEMATOLOGY

HISTORY, PHYSICAL EXAMINATION & LAB TESTS

SIGNS & SYMPTOMS

History

Signs & symptoms

Clinical signs

Signs & symptoms of anemia

Signs & symptoms of cytopenia

Lab tests

CBC (COMPLETE BLOOD COUNT) PARAMETERS / PERIPHERAL BLOOD SMEAR

CBC (complete blood count)

Characteristics that may affect CBC test results

Causes for rejection

Red blood cell (RBC) count

RΒC count – limitations

Hemoglobin (Hb; Hgb)

Causes of low and high Hemoglobin (Hb; Hgb)

Hb – limitations

PCV (packed cell volume)

Hematocrit (Ht; HCT)

The relationship between hemoglobin & hematocrit values and when a mismatch may occur  

Causes of decreased & increased Hematocrit (Ht; Hct)

Hematocrit – limitations

Mean corpuscular/cell volume (MCV)

Mean corpuscular/cell – hemoglobin (MCH)

Mean corpuscular/cell hemoglobin concentration (MCHC)

Reticulocyte count

Absolute reticulocyte count

Leukemoid reaction

Leucoerythroblastic reaction

Leukopenia

EFFECTS OF HEMOLYSIS & LIPEMIA ON CLINICAL SPECIMENS

Hemolysis on clinical specimens

Effects of hemolysis on clinical samples

Hemolysis & blood clotting studies

Prevention of hemolysis on clinical specimens

Advice to prevent hemolysis during venipuncture

LIPEMIC SPECIMENS

    INTRODUCTION IN HEMATOLOGY

    HISTORY, PHYSICAL EXAMINATION & LAB TESTS

    SIGNS & SYMPTOMS

         History

·         Medical history (present, past – including childhood & infancy)

·         Medication (including over-the-counter drugs, herbs, dietary supplements, the Pill (contraceptives), and HRT (hormone replacement therapy))

·         Occupation, hobbies

·         Administration of corticosteroids, GM – CSF?

·         Bleeding diathesis (e.g., after minor surgery, e.g., at the dentist)

·         Smoker? If yes, heavy?

·         Alcohol abuse?

·         Hospital admissions?

·         Surgery?

·         Pap test (smear) & mammogram for women, PSA for men?

·         Pets, contact with animals (including birds)?

·         Transfusion? If yes, were there any reactions?

·         Family history (e.g., bleeding problems).

·         Other family or friend/ colleague with similar symptoms

       Signs & symptoms

·         Symptoms of viral disease?

·         Travel to a foreign country?

·         Weight loss?

·         Anorexia?

·         Fatigue?

·         Itching?

·         Dyspnoea (SOB (Shortness of breath))?

·         Pruritus (itchiness) after a warm bath (polycythemia vera)?

·         Nocturnal (night) sweats (lymphoma, TB (tuberculosis))?

·         Bleeding on dentist/ minor surgery?

·         Pain following alcohol consumption (a sign of Hodgkin lymphoma)

·         Easy bleeding and petechiae due to low platelet count?

·         Lassitude (fatigue)?

·         Clinical signs:

·         Splenomegaly/ hepatomegaly/ hepatosplenomegaly?

·         Lymphadenopathy (enlarged lymph nodes)?

·         Fever?

·         Bleeding diathesis?

·         Gum enlargement and/or bleeding?

·      Signs & symptoms of anemia: 
   Pallor? Palpitations? Flow murmurs? Tiredness, Dyspnoea (SOB (Shortness of breath))?  Angina? Signs of heart failure?

·         Signs & symptoms of cytopenia:

·         a) Red blood cells: anemia [pallor, fatigue, weakness, paleness, SOB (shortness of breath], palpitations (may have flow murmurs), angina & signs of heart failure may occur].

·         b) White blood cells: leukopenia (fever, infections).

·         c) Platelets: thrombocytopenia (bleeding tendencies, petechia; easy bruising, bleeding with minor trauma).

       Lab tests:

·         Biochemistry: potassium, sodium, LDH (may be increased in various hematological problems), calcium (corrected or ionized), phosphorus, glucose, BUN (blood urea nitrogen), creatinine, liver function tests (ASL, ALT, GGT), alkaline phosphatase, CPK (CK), bilirubin (total, direct, indirect), iron, ferritin, TIBC, vitamin B12 & folate [megaloblastic anemia (B12 and/or folate deficiency, pernicious anemia may be followed by pancytopenia], CRP

·         + IgM, IgG, IgE, IgA

·         + Serum & urine protein electrophoresis

·         Hematology: FBC (full blood count, also known as CBC (complete blood count)), reticulocyte count, ESR

·         + Peripheral blood smear

·         + Blood thin & thick films

·         Serology: virology (EBV, CMV, HIV, HSV, VZV, parvovirus B19, HAV, HBV, HCV, human herpesvirus-8 (HHV8, causes sarcoma Kaposi), tests for toxoplasma, Leishmaniasis, (may cause pancytopenia), rickettsia (tick bite?), brucellosis, typhoid

·         Mayer (stool hemoglobin)

·         +_Tumor markers

·         +_Mantoux (for TB)

                          

·         Imaging tests:

·         Abdominal ultrasound (splenomegaly? Hepatomegaly?)

·         CXR (chest X-rays)

·         +_Colonoscopy & gastroscopy

·         +_CT

CBC (COMPLETE BLOOD COUNT) PARAMETERS/ PERIPHERAL BLOOD SMEAR

  

    CBC (COMPLETE BLOOD COUNT) PARAMETERS

· CBC (complete blood count; also called FBC (full blood count)): normal/increased/decreased: WBCs (white blood cells (check ALWAYS the absolute number of each type of white blood cell!)), RBCs (red blood cells), MCV, MCH, MCHC, RDW (red blood cell distribution width; shows the variation in the cellular volume of the RBCs), platelets (Plts), MPV (mean platelet volume; measurement of the average size of the Plts); +_reticulocyte production index (often very useful).

·         Specimen: on EDTA whole blood.

·      Characteristics that may affect CBC test results: lipemia, bilirubinemia (jaundice), cold agglutinins, warm agglutinins, hemolysis, electrolyte imbalances, and WBC fragments. Marked changes in plasma constituents (e.g., low sodium, extremely elevated glucose) may cause cells to swell or shrink. The blood-to-anticoagulant ratio is essential.

·    Causes for rejection: clotted samples; grossly hemolyzed samples; samples drawn from above an intravenous line (IV); specimens other than EDTA whole blood; improper labeling; samples not stored properly; samples older than stability limits.

·   Red blood cell (RBC) count: a rise or drop in the RBC count must be interpreted in conjunction with other parameters, such as hemoglobin, hematocrit, reticulocyte count, and/or red blood cell indices. Blood or red cell loss that occurs suddenly or over time and diseases and conditions that decrease red blood cell production in the bone marrow will result in a low RBC count.

·         Women tend to have slightly lower RBC counts than men.

·         A recent blood transfusion can affect the results of an RBC count.

·         Alteration of the number of RBCs is often temporary and can be easily corrected and/or returned to normal levels by treating and resolving the underlying condition.

·         During pregnancy, body fluids tend to accumulate, thus decreasing the RBC count related to the fluid volume.

·         Living at high altitudes causes an increase in RBC count; this is the body's response to the decreased oxygen available at these heights.

·         Some causes of a low RBC count (anemia) include:

·         a) Trauma.

·         b) Red blood cell destruction, e.g., hemolytic anemia caused by autoimmunity or genetic defects in the red cell itself. Errors on the red blood cells include hemoglobinopathy (sickle cell anemia or thalassemia), an abnormality in the RBC membrane (e.g., hereditary spherocytosis), or enzyme defect (e.g., G6PD deficiency).

·         c) Acute or chronic bleeding from the digestive tract (e.g., peptic ulcers, polyps, colon cancer) or other sites, such as the bladder or uterus (e.g., in women, heavy menstrual bleeding).

·         d) Nutritional deficiency such as iron, vitamin B12, or folate deficiency.

·         e) Bone marrow damage (e.g., toxin, radiation or chemotherapy, infection, drugs).

·         f) Bone marrow disorders such as leukemia, multiple myeloma, myelodysplasia (MDS), or lymphoma, or other cancers that spread to the marrow

·         g) Chronic inflammatory disease or condition.

·         h) Kidney failure. Severe kidney disease and chronic kidney disease (CKD) lead to decreased production of erythropoietin (EPO; a hormone produced by the kidneys that stimulates RBC production by the bone marrow).

·         i) Cold agglutinin disease. 

·         Some causes of a high RBC count (polycythemia) include:

·         a) Dehydration. As the volume of fluid in the blood drops, the number of RBCs per volume of fluid artificially rises.

·         b) Pulmonary disease. In patients with dyspnea from lung disease, the body tries to compensate by producing more red blood cells. Pulmonary causes include pulmonary hypertension, COPD, and hypoventilation syndromes.

·         c) Sleep apnea.

·         d) Congenital heart disease. With this condition, the heart is not able to pump blood efficiently, resulting in a decreased amount of oxygen getting to tissues. The body tries to compensate by producing more red blood cells.

·         e) Congestive heart failure (CHF) and abnormal flow of blood from the right side to the left side of the heart.

·         f) Kidney tumor that produces excess erythropoietin. Also, hepatocellular carcinoma, adrenal adenoma, and uterine tumors.

·         g) Smoking.

·         h) Kidney cysts and hydronephrosis. Also, renal hypoxia, kidney transplantation, and renal artery stenosis.

·         i) Genetic causes (altered oxygen sensing, abnormality in hemoglobin oxygen release) including 2,3 – BPG (higher oxygen affinity by hemoglobin).

·         j) Chuvash polycythemia (congenital).

·         k) Polycythemia vera (a rare disease in which the body produces excess RBCs inappropriately).

·       RΒC count – limitations: cold agglutinins and rare warm agglutinins cause decreased RBC counts.

·         Increased white cell counts may cause falsely elevated RBC counts.

·         A recent blood transfusion and pregnancy can affect red blood cell counts. People who live at higher altitudes may have higher red blood cell counts, and women have lower red blood cell counts than men.

·     Hemoglobin (Hb; Hgb): used to screen for and help diagnose conditions that affect red blood cells (RBCs); anemia (low hemoglobin) or polycythemia (high hemoglobin), to assess the severity of these conditions and to monitor response to treatment.

·         The clinical definition of anemia is related to either an abnormal Hematocrit (Hct) or Haemoglobin (Hgb) value.

·         Reference range:

·         a) Men: 14.0 – 17.5 (mean 15.7) g/dL.

·         b) Women: 12.3 – 15.3 (mean 13.8) g/dL.

·         For reference range for children, see:

·         http://emedicine.medscape.com/article/2085614-overview#a1

·         Causes of low and high Hemoglobin (Hb; Hgb):

·         Low hemoglobin – interpretation: Hb is used clinically to determine the presence of anemia, which is functionally defined as insufficient red blood cell (RBC) mass to adequately deliver oxygen to peripheral tissues.  Anemia is considered to be present if the Hb or the hematocrit (Hct) is below the lower limit of 2 standard deviations (-2SD) or the 95% confidence interval for the average population. This definition of anemia results in 2.5% of healthy individuals being classified as anemic. Anemia is absolute if the RBC mass is decreased and relative if associated with an increased plasma volume. 

·         Absolute anemia can be divided into 2 main categories: decreased RBC production and high RBC destruction or loss over the bone marrow’s ability to replace those losses.

·         Causes of decreased RBC production include nutritional deficiencies (iron, folate, vitamin B12, vitamin B6), anemia of chronic disease, renal, liver, or endocrine disease, bone marrow infiltration (myelophthisic anemia), aplastic anemia, pure red cell aplasia, and sideroblastic anemia.

·         Causes of increased RBC destruction or loss over the bone marrow’s ability to replace those losses include blood loss (hemorrhage), hemolysis of various etiologies (both intrinsic and extrinsic), and hemoglobin disorders (hemoglobinopathies and thalassemias). 

·   Relative anemia, on the other hand, may be seen in overhydration (volume overload), pregnancy, macroglobulinemia, and post-flight astronauts.  

·     High hemoglobin – interpretation: higher-than-normal Hb may be indicative of polycythemia. The World Health Organization (WHO) classification of hematologic malignancies defines polycythemia as Hb greater than 18.5 g/dL in men, higher than 16.5 g/dL in women, Hct greater than 99th percentile of the method-specific reference range, or Hb greater than 17 g/dL in men, higher than 15 g/dL in women with a documented and sustained increase of at least 2 g/dL from baseline not attributed to correction of iron deficiency. 

·         Absolute polycythemia is an increase in the total RBC mass in the body. Causes include hypoxia, inappropriate erythropoietin production, genetic polycythemia, and polycythemia vera.

·         Relative polycythemia is an increase in Hct or RBC count resulting from a decrease in plasma volume; however, total RBC mass is not increased. Causes include dehydration, shock, diuretic therapy, and spurious polycythemia (Gaisbock syndrome). Note: Gaisbock syndrome is a specific type of relative polycythemia primarily occurring in obese men in whom hypertension causes a reduction in plasma volume, resulting in (amongst other changes) a corresponding increase in red blood cell count.

·         Hemoglobin (Hb) – limitations:
A remarkably elevated white blood cell count may cause an increased hemoglobin value.

·         Abnormal paraproteins found in multiple myeloma patients, lipemia, or severely icteric samples can falsely increase hemoglobin results.

·         Hemoglobin S, C, and F, or hereditary or acquired spherocytosis may cause an increase in hemoglobin values.

·         Removal of plasma from a sample will produce falsely elevated hemoglobin results.

·         Also, recent transfusions can affect hemoglobin levels. Additionally, they are slightly lower during pregnancy.

·    PCV (packed cell volume): a known volume of anticoagulated whole blood is spun in a centrifuge tube, and the volume occupied by the red cell is measured.

·     Hematocrit (Ht; HCT): an indirect measure of PCV computed by hematology cell analyzers from red cell count and mean volume, i.e., Hematocrit = mean cell volume (x) Red cell count.

·         The clinical definition of anemia is related to either an abnormal Hematocrit (Hct) or Hemoglobin (Hgb) value.

·         Reference range: it varies depending on the methodology used. Normal ranges should be validated by individual clinical laboratories. Reference ranges (SI units/conventional units):

·         a) Males: 0.40 – 0.54 (40-54%).

·         b) Females: 0.36 – 0.46 (36-46%).

·         c) Newborns: 0.53 – 0.69 (53-69%).

·         The relationship between hemoglobin & hematocrit and when a mismatch may occur

·         The Hematocrit (Hct) (%) is usually defined as three times the value of hemoglobin (Hgb) (g/dl).

·      In cold agglutinin disease, there may be a mismatch between hemoglobin & hematocrit values.

·    Cold agglutinins are antibodies that are usually specific for I antigen, an RBC surface carbohydrate macromolecule. Cold agglutinins bind to the erythrocyte surface antigen at a temperature optimum of 0 – 4 °C, which causes agglutination of erythrocytes and, thereby, impaired microcirculation ranging from moderate acrocyanosis to severe Raynaud’s phenomenon. The antigen-antibody complex also induces the classical complement pathway, resulting in extravascular hemolysis. The physiologic cooling of blood in peripheral vessels is usually sufficient to cause hemolysis and circulatory symptoms in patients with a high thermal amplitude of cold agglutinins. The clinical effects of cold agglutinins depend on the antibody's titer and thermal amplitude. Cold agglutinins are rare, and only low titers can be found in the serum of healthy individuals. Cold agglutinins may be monoclonal or polyclonal. Monoclonal antibodies are usually detected in patients with idiopathic forms of cold agglutinin disease or lymphoproliferative disorders. Polyclonal antibodies typically appear after infection, most often with Mycoplasma pneumonia, EBV, or CMV. Cold agglutinins can cause hemolysis in patients undergoing cardiac surgery during a hypothermic cardiopulmonary bypass. In cold agglutinins, Hematocrit may appear to decrease, and MCV elevated (Reference: http://www.biochemia-medica.com/2014/24/391 ).

·         If the cold agglutinin is operative at room temperature, then a falsely high mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) with a low RBC count is obtained due to agglutination of RBCs in the cold, automated counter.

·         Agglutination may also be seen in anticoagulated blood at room temperature. This agglutination worsens with storage and cooling of blood to 4°C and disappears rapidly upon warming the blood to 37°C, unlike with rouleaux formation. Repeating the complete blood count (CBC) after heating the blood to 37°C avoids this problem. Thus, the clinical laboratory is frequently the first to report the presence of a cold agglutinin.

·         A study concluded that cold agglutinins may interfere with the analysis of erythrocyte and erythrocyte-related parameters (Hematocrit, MCV, MCH, and MCHC); however, Hb, leukocyte, and platelet counts are not affected (Reference: http://www.biochemia-medica.com/2014/24/391 ).

·         The combination of low hematocrit, normal hemoglobin, and high MCV and MCHC are characteristic of cold agglutinins.

·         Often, the first indication of the presence of cold agglutinins is the failure to obtain a meaningful RBC count and indices. The hemoglobin and hematocrit results do not match. The RBC count will be decreased due to doublet erythrocytes being counted as a single cell, thus resulting in a falsely high MCV. Hematocrit will also be lowered, as the volume of doublets is slightly less than 2 cells. The MCHC and MCH values will be increased due to decreased hematocrit and RBC count. Invalid red blood cell indices can also be due to lipemia, hemolysis, icterus, or hereditary spherocytosis (Reference: http://labmed.oxfordjournals.org/content/labmed/33/6/455.full.pdf ).

·      Causes of decreased & increased Hematocrit (Ht; Hct):

·         Main causes of reduced hematocrit include:

·         a) Anemia, e.g., iron deficiency anemia, aplastic anemia, thalassemia, lead poisoning, etc.

·         b) Bleeding, e.g., acute blood loss from trauma (Ht may be normal initially), gastrointestinal bleeding, etc.

·         c) Red blood cell destruction, e.g., autoimmune hemolytic anemia, drug-induced hemolytic anemia, thrombotic thrombocytopenic purpura (TTP), sickle cell disease, splenomegaly, etc.

·         d) Bone marrow suppression or underproduction, e.g., leukemia, myelodysplasia, chemotherapy, etc.

·   e) Malnutrition and nutritional deficiencies, e.g., folate deficiency, vitamin B12 deficiency, Kwashiorkor (a form of severe protein-energy malnutrition characterized by edema, irritability, anorexia, ulcerating dermatoses, and an enlarged liver with fatty infiltrates), etc.

·         f) Infection, e.g., parvovirus, sepsis, etc. 

·        g) Overhydration, e.g., polydipsia, etc.

·         h) Pregnancy.

·         Main causes of increased hematocrit include:

·         a) Dehydration, e.g., from fluid suppression, heat stroke, etc.

·         b) Congenital heart disease.

·         c) Cor pulmonale, e.g., sleep apnea, chronic obstructive pulmonary disease (COPD), etc.

·         d) Erythrocytosis, e.g., primary or secondary polycythemia vera, etc.

·         e) Hypoxia in low-oxygen states, e.g., high altitude, premature babies with delayed lung development, etc.

·   Hematocrit – limitations: cold agglutinins and rare warm agglutinins may cause decreased hematocrit values. Also, hematocrit results can be affected by recent transfusions and during pregnancy.

·    Mean corpuscular/cell volume (MCV) in fl (femtoliters) = Hct (%)/ RBC count. Normal range: 80 – 100 fl. Normocytic, microcytic, macrocytic.

·         Mean corpuscular/cell – hemoglobin (MCH) in pg (picograms) = (Hb/ RBC count) x10. Normal range: 27 – 31 pg. Hupochromic, normochromic.

·         Cold agglutinin disease may cause increased MCH.

·         Mean corpuscular/cell hemoglobin concentration (MCHC) in g/dL (picograms) = MCH/MCV.  Normal range: 32 – 36 g/dL. It is lowered (hypochromic) in microcytic anemias but is normal (normochromic) in macrocytic anemias (due to larger cell size, although the hemoglobin amount (MCH) is high, the concentration remains normal). It is increased in hereditary spherocytosis, sickle cell disease, and homozygous hemoglobin C disease. A very high MCHC (>370 g/L) may indicate blood with cold agglutinin.

·         Reticulocyte count: (Reticulocytes counted/ number of RBCs) x 100. Normal value: 0.5% – 2.5%. Reticular Index (corrected reticulocyte count) = reticulocytes counted x Hematocrit (%)/ average Hematocrit for age (normal hematocrit). A value of 45 is usually used as normal hematocrit. Reticular Production Index = Reticular Index/maturation correction; the other formula is reticular count x (observed hemoglobin/ normal hemoglobin) x 0.5. Maturation correction: for Ht 36 – 45 is 1; for Ht 26 – 35 is 1.5; for Ht 16 – 25 is 2; and for Ht <_15 is 2.5. 

·         Absolute reticulocyte count = reticulocyte percentage x RBCs count.

·         Leukemoid reaction: WBC (white blood cells) > 50 000/ cmm and immature WBCs in peripheral blood.

·         Myeloid leukemoid reaction is characterized by marked neutrophilic leucocytosis with the presence of premature WBCs (white blood cells); it mimics CML (chronic myelogenous leukemia). Causes: severe bacterial infection, severe hemolysis or hemorrhage, metastasis to bone marrow, burns, eclampsia.

·       A lymphoid leukemoid reaction is characterized by marked reactive lymphocytosis; it may resemble ALL (acute lymphocytic leukemia) or CLL (chronic lymphocytic leukemia). Causes: viral infections (e.g., infectious mononucleosis and infectious lymphocytosis) and bacterial infections (TB (tuberculosis), whooping cough (pertussis)).

·         Leukemoid reaction – Peripheral blood smear: a mix of early mature neutrophil precursors, contrary to immature forms seen in acute leukemia. Serum leukocytes' alkaline phosphatase (ALP) is normal or elevated, contrary to CML (chronic myelogenous leukemia), where it decreases.

·         Leukemoid reaction – Causes (generally): hemorrhage, drugs (sulfa drugs, dapsone, glucocorticoids, G – CSF and related growth factors, all-trans retinoic acid), infections (TB (tuberculosis), pertussis, Clostridium dificille, infectious mononucleosis (here lymphocytes predominate), visceral larva migrans (here eosinophils predominate), asplenia, DKA (diabetic ketoacidosis), hepatic necrosis, ischemic colitis, trisomy 21 (Down’s syndrome) in infancy (10%), and paraneoplastic phenomenon (rare).

·         Leucoerythroblastic reaction: the presence of immature WBCs as well as nucleated RBCs (red blood cells) in peripheral blood. Causes: infections (e.g., miliary tuberculosis), metastatic cancers to the marrow, myelofibrosis, severe hemolysis, lymphoma, myeloma, storage disorders (Gaucher’s disease, Niemann Pick disease), osteopetrosis.

·         Leukopenia: decreased in the number of WBCs (white blood cells). Increased risk of infection. Neutropenia is reduced in the number of circulating neutrophil granulocytes.

·         Causes of leukopenia: chemotherapy, radiation therapy, myelofibrosis, aplastic anemia, viruses (e.g. HIV), SLE (systemic lupus erythematosus), lymphoma Hodgkin’s, folate deficiency, infections (Lyme disease, rickettsia infections, dengue, leishmaniasis, typhoid, malaria, tuberculosis, dengue, etc), sepsis, some types of cancer, spleen enlargement, copper and zinc deficiency, arsenic toxicity, corticosteroids administration (however they may cause neutrophilia), liver cirrhosis, etc. 

·         Pseudoleukopenia: may develop on the onset of an infection as neutrophils migrate towards the site of the infection.

·         Drugs that may cause leukopenia: clozapine (an antipsychotic), bupropion (for smoking and depression), antiepileptics (valproic acid & lamotrigine), immunosuppressive medications, interferons (for multiple sclerosis), and cancer chemotherapy. 

      EFFECTS OF HEMOLYSIS & LIPEMIA ON CLINICAL SPECIMENS

       Hemolysis on clinical specimens

·         Hemolysis due to the breakdown of red blood cells is essential to the laboratory because it can influence laboratory results. The effects can result from products liberated from the red cells themselves or interferences with laboratory analyzers. This may vary from one test to another depending on the formulation of the reagent.

·         Hemolysis can occur in vivo (in the patient) due to a variety of medical conditions, including antigen-antibody reactions, hemolytic anemias (including autoimmune hemolytic anemia), transfusion reaction, DIC, severe infections, toxins and poisons, mechanical RBC rupture due to artificial heart valves, as well as treatments such as hemodialysis and the use of the heart-lung bypass machine. 

·         Hemolysis can occur during suboptimal blood collection or in vitro (e.g., in the collection tube) due to improper handling, transport, and storage.

·         Hemolysis can be recognized in the laboratory by a visual inspection of the plasma or serum sample, which appears rosy to bright red in color.

·         Samples with slight hemolysis are analyzed, and the results are reported with a comment indicating the degree of hemolysis and the effect on the test result.

·         Grossly hemolyzed samples can affect the results of many tests; therefore, a recollection will be requested for most grossly hemolyzed specimens.

         Effects of hemolysis on clinical specimens

·         Results from all laboratory disciplines can be affected by hemolysis, especially in chemistry. Some of the more routine hematology tests involved are haptoglobin, lactate dehydrogenase (LD; LDH), folate, and iron.

·         The amount of hemolysis needed to affect a test is dependent on the test being performed. Generally, slight hemolysis has little effect on most tests; however, it will cause increased test results for specific tests like potassium (K⁺) and lactate dehydrogenase (LD; LDH).

·         Effects of hemolysis on hematological parameters results:

·         a) Slight change:

·         i) Test result decreased by hemolysis: haptoglobin, bilirubin.

·         b) Noticeable difference: Test result increased by hemolysis: iron, coagulation tests. 

·         c) Significant change:

·         i) Test results increased by hemolysis: potassium (K⁺), lactate dehydrogenase (LDH; LD), AST (SGOT).

·         ii) Test results increased or decreased by hemolysis: (HGB) hemoglobin, RBCs (red blood cells count), MCHC, Platelet count.

·         Lipemia, hyperbilirubinemia, and hemolysis interfere with the detection of clot formation by photo-optical methods and cause falsely elevated values on the results of the PT (prothrombin time).

·         Overview of effects on hematological parameters results:

·         a) Moderate effects: lactate dehydrogenase (LDH), potassium (K⁺), bilirubin, FVIIa (activated factor VII), PT (prothrombin time), CEPI, CADP. 

·         b) It may also affect the results of D-Dimers and aPTT. 

·         In a study, sixteen (16) healthy volunteers were enrolled. Four hemolysis levels were constituted according to hemoglobin concentrations, and they were divided into five groups: Group I: 0 – 0.10 g/L, Group II: 0.10 – 0.50 g/L, Group III: 0.51 – 1.00 g/L, Group IV: 1.01 – 2.50 g/L, Group V: 2.51-4.50 g/L. Lysis was achieved by mechanical trauma. Hemolysis interference affected lactate dehydrogenase (LD; LDH) and aspartate aminotransferase (AST; SGOT) almost at undetectable hemolysis by visual inspection (plasma hemoglobin < 0.5 g/L). Clinically meaningful potassium and total bilirubin variations were observed in moderately hemolyzed samples (hemoglobin > 1 g/L). Alanine aminotransferase (ALT; sGPT), cholesterol, gamma-glutamyltransferase (GGT), and inorganic phosphate (P) concentrations were not interfered up to severely hemolyzed levels (hemoglobin: 2.5 – 4.5 g/L). Albumin, alkaline phosphatase (ALP), amylase, chloride, HDL-cholesterol, creatine kinase (CK), glucose, magnesium, total protein, triglycerides, unsaturated iron-binding capacity (UIBC), and uric acid differences were statistically significant but remained within the CLIA limits. The study concluded that to avoid preanalytical visual inspection for hemolysis detection, improper sample rejection, and/or rerun because of hemolysis, it is recommended in this study that routine determination of plasma or serum-free hemoglobin concentrations is essential. For hemolysis, new samples must be requested.

·         In another study, nine aliquots were tested for the most common clinical chemistry analytes, prepared by serial dilutions of homologous hemolyzed samples collected from 12 different subjects and containing a final concentration of serum hemoglobin ranging from 0 to 20.6 g/L. Lysis was achieved by subjecting whole blood to an overnight freeze-thaw cycle. Hemolysis interference appeared to be approximately linearly dependent on the specimen's final concentration of blood-cell lysate. This generated a consistent trend towards overestimation of alanine aminotransferase (ALT; SGPT), aspartate aminotransferase (AST; SGOT), creatinine, creatine kinase (CK), iron, lactate dehydrogenase (LD; LDH), lipase, magnesium, phosphorus, potassium, and urea, whereas mean values of albumin, alkaline phosphatase (ALP), chloride, gamma-glutamyltransferase (GGT), glucose and sodium were substantially decreased. Clinically meaningful variations of AST (SGOT), chloride, lactate dehydrogenase (LD; LDH), potassium (K⁺), and sodium (Na⁺) were observed in specimens displaying mild or almost undetectable hemolysis by visual inspection (serum hemoglobin < 0.6 g/L). The varied and unpredictable response to hemolysis observed for several parameters prevented the adoption of reliable statistical corrective measures for results by the degree of hemolysis. The study concluded that if hemolysis and blood cell lysis result from an in vitro cause, the authors suggest that the most convenient corrective solution might be a quantification of free hemoglobin, alerting the clinicians and sample recollection.

·     A 1992 study examined the effects of hemolysis on the results of twenty-five common biochemical tests. The scientists collected sixty blood samples of 15mL from inpatients and outpatients and mechanically hemolyzed 10 mL of the samples in a two-step procedure. They classified serum from these samples as being not hemolyzed, moderately hemolyzed, or severely hemolyzed and then performed 25 common biochemical tests. Statistical analysis of the results showed that hemolysis had the most significant effect on the lactate dehydrogenase (LD; LDH), acid phosphatase, and potassium (K⁺) tests.

   
       Hemolysis & blood clotting studies

·         Lipemia, hyperbilirubinemia, and hemolysis interfere with the detection of clot formation by photo-optical methods and cause falsely elevated values on the results of the PT (prothrombin time).

·         The Clinical and Laboratory Standards Institute guidelines for prothrombin time (PT) and activated partial thromboplastin time (aPTT) testing states: "Samples with visible hemolysis should not be used because of possible clotting, factor activation and interference with endpoint measurement.

·         Hemolysis increases the spectrometric absorbance of the plasma sample and leads to high background absorbance readings, which may compromise clot detection by some instruments and thus affect the accuracy of test times. Devices utilizing mechanical means of clot detection are not affected by this interference, but the test result may still be compromised since cell lysis products include tissue factors that may activate coagulation. The net effect is that detected fibrinogen levels may fall with increasing hemolysis, whereas D-dimer levels may rise. Prothrombin time values may fall in line with decreasing fibrinogen, whereas APTTs may increase or decrease depending on the net effect of activation vs. the loss of fibrinogen. Hemolysis may also influence other test results (e.g., decreased antithrombin levels).

·         If possible, grossly hemolyzed specimens should be rejected. If testing must be pursued (e.g., if in vivo hemolysis is present), measurement using a mechanical endpoint detection system is recommended, although the potential effect of activation should also be noted. Samples appearing hemolyzed due to the presence of a hemoglobin substitute are not a cause of specimen rejection, and these samples should be evaluated using a mechanical or electromechanical method for clot detection.

        Prevention of hemolysis on clinical specimens

·         a) Vein size and trauma. Puncturing small, fragile veins and probing or “fishing” the vein with a needle can lead to hemolysis. The phlebotomist should choose an appropriately sized vein and use phlebotomy equipment suitable for the vein size. If the vein is fragile, he/she should not use large-volume tubes. If a vein is traumatized during puncture, the first tube collected may be hemolyzed, while subsequent tubes are fine. The phlebotomist should avoid puncturing areas that have a hematoma.

·         b) Alcohol preparation. The phlebotomist should allow the alcohol to dry completely before venipuncture. The needle can transfer wet alcohol from the skin into the blood specimen and cause hemolysis.

·         c) Needle size. Using a large needle (larger bore=lower gauge) can cause hemolysis by allowing a large amount of blood to suddenly enter the tube with great force. Similarly, the use of needles that are too fine (higher gauge) can also cause hemolysis by forcing the blood through a tiny opening under great force. The red cell walls become sheared on the needle as they enter the tube.

·         d) Loose connections. The phlebotomist should ensure that all connections of the collection components are tightened, i.e., the connections between a blood collection set and luer adapter, between the syringe and needle, and between the catheter and luer adapter. Loose connections introduce air into the system and cause frothing in the specimen, which can result in hemolysis.

·         e) Underfilled Tubes. The phlebotomist should properly fill all tubes to ensure the proper blood-to-additive ratio. Certain additives in high concentrations, such as sodium fluoride, can cause varying degrees of hemolysis.

·         f) Syringe collections. Improper syringe draws are notorious for causing hemolyzed specimens. If possible, syringe use should be avoided in favor of the evacuated tube system. A study evaluated the effects of specimen quality when using syringe draws compared to the evacuated tube system. Visual hemolysis was found in 19% of specimens drawn by syringe, compared to 3% when drawn by the evacuated tube system. Also, syringe-collected samples exhibited clotted EDTA specimens in 11% of the patients, as opposed to none in the evacuated tube system. If a syringe must be used, the following recommendations can reduce the incidence of hemolysis:

·     1) The phlebotomist should pump the plunger 2 – 3 times before collection to loosen the plunger. Then the phlebotomist should tighten the needle and syringe connection.

·         2) The phlebotomist should use a 3 –10mL syringe, avoiding larger volumes if possible.

·       3) The phlebotomist should ensure that the aspiration speed does not exceed 1mL of air space during collection. Excessive aspiration forces frequently cause hemolysis.

·         4) The phlebotomist should perform blood transfer into the tube immediately.

·         5) The phlebotomist should fill the tube by vacuum only but never push down on the plunger, as this increases the force of the blood flow, creating a high degree of red blood cell (RBC) trauma. More importantly, positive pressure is produced in the tube, with the potential to cause either tube breakage or stoppers to pop out.

·         6 The phlebotomist should use a blood transfer device to transfer syringe-collected blood into a tube. It will enhance safety and improve specimen quality.

·         7) The phlebotomist should angle the syringe so that the blood runs down the side of the tube. By preventing the cells from hitting the bottom of the tube with such a great force, RBCs (red blood cells) trauma can be reduced.

·         g) Peripheral Catheter Collections. The highest rates of hemolyzed specimens appear to come from the acute care setting, i.e., Emergency Dept (ED), Labor and Delivery (L&D), and Intensive Care Units (ICU). Studies have shown that the main source of hemolysis in the ER (emergency room) is the use of IV catheters for specimen collection. One study found that specimens drawn by nurses through an IV catheter were more than 3 times as likely to be hemolyzed than those drawn by venipuncture (13.7% vs. 3.8%). In another study, specimens were collected using IV catheters and venipuncture and then compared. The results revealed a 50% rate of hemolysis in the IV catheter collected specimens, compared to no hemolysis at all in the [peripheral] venipuncture specimens. Blood collected from the back of the IV catheter is pulled through several gauges; the catheter ranges from 18 to 22G, the Luer adapter “front” end is 15G, and the stopper-piercing needle is 20G. Slowing down this “pull rate” can reduce the hemolysis rate significantly. The use of partial draw collection tubes is an effective way to slow down the pressure exerted on the blood and, thus, reduce hemolysis. The reduced vacuum in these tubes yields a slower, gentler draw. Partial drawtubes are designed to fill “part way” while maintaining the proper blood-to-additive ratio. The smaller volume of blood drawn into partial-draw tubes satisfies the CAP recommendation for minimizing large blood draw volumes and mitigates safety concerns as less blood is handled and discarded (partial-draw tubes should not be confused with small-size pediatric tubes that are fully evacuated).

·         h) Specimen Handling Techniques. Once the proper specimen collection techniques are applied, subsequent specimen handling factors must be considered to prevent hemolysis from occurring in the pre-analytical phase. The following factors should be considered to prevent hemolysis:

·         1) Mixing Tubes. The phlebotomist should mix the blood with the tube additive through gentle tube inversions. The phlebotomist should not shake the tube after collection.

·         2) Transport Methods. The phlebotomist should be cautious with pneumatic tube systems and other rough transport conditions that can create turbulence and RBC trauma within the tube. The phlebotomist should hand deliver specimens when feasible. Specimens should be stored in an upright position following centrifugation.

·         3) Rimming clots. The phlebotomist should not use wooden applicator sticks to rim clots, which can shred the red cells. With the currently evacuated serum tubes available, rimming clots is unnecessary.

·         4) Temperature. The lab technicians should store and transport specimens in controlled temperature conditions, as temperatures that are too high or too low can rupture red cell membranes. They should make sure that centrifuge temperatures are acceptable. They should abide by the recommended transport and storage temperatures specified by the laboratory that is performing the assays.

·         i) Centrifugation. Optimally, serum should be separated from the clot by centrifugation within one-half hour after collection. This not only maintains the sample integrity but will also prevent the sample from hemolysis after venipuncture.

     Advice to prevent hemolysis during venipuncture

·         The venipuncture site should be allowed to dry after cleansing

·         The phlebotomist should use the largest bore needle that’s appropriate

·         The phlebotomist should never draw blood through a hematoma

·         The phlebotomist should remove the tourniquet as early as possible to decrease flow velocity and turbulence

·         The phlebotomist should not remove the collection tube until full

·         If using a syringe, the phlebotomist should ensure the needle is fitted securely to avoid frothing

·         If using a syringe, the phlebotomist should avoid drawing the plunger back too forcibly

·         When mixing is required, gentle inversion is adequate.

·         See also:

·         https://www.ena.org/practice-research/research/CPG/Documents/HemolysisSynopsis.pdf

           Lipemic specimens

·         Lipemic plasma has large lipid particles that include lipoproteins and chylomicrons. As a result, these samples have increased sample turbidity and may prolong coagulation results. Interference is variable among analyzers. Turbid samples cause attenuation of the intensity of light passed through a sample due to scattering, reflectance, or absorption.

·         Lipemia and hyperbilirubinemia interfere with the detection of clot formation by photo-optical methods. The results of the PT (prothrombin time) may be affected.

·         Lipemia, hyperbilirubinemia, and hemolysis interfere with the detection of clot formation by photo-optical methods and cause falsely elevated values on the results of the PT (prothrombin time).