Overview on Ordering and Evaluation of Laboratory Tests

Etiology and Epidemiology

Laboratory investigations play a pivotal role in diagnosing and management of diseases. The number of tests ordered daily rises with the number and complexity of diagnoses at discharge. Patients with a single diagnosis were found to undergo 7.5 tests per day. A study observed that the daily average for tests in the internal medicine department was 9.5. Similar results were found by another study performed at an American hospital stating an average of 9.67 tests per inpatient day. The average for CBC (complete blood count), an important initial test frequently ordered, was 1.07. The most frequently ordered analytes were serum electrolytes levels, particularly sodium.[3][4]

Specimen Requirements and Procedure

Blood Specimen

After identifying the patient, a phlebotomist takes a blood sample in a syringe from an appropriate vein (preferably antecubital). Blood is collected in properly labeled containers. Serological tests are performed on a clotted blood specimen. It should be protected from extreme temperatures during transport and refrigerated at -20 degrees celsius or lower and sent frozen to the laboratory. A serum for serology, on the other hand, is kept at 2 to 8 degrees celsius.

For blood cultures, contact the laboratory staff for appropriate media, as it depends upon the type of pathogens. Change the syringe needle before injecting blood into the culture bottle. The rubber bung of the culture bottle needs to be cleaned with iodine solution before and after injecting blood into it. The quantity of blood injected is 10% of the liquid medium volume (for 30 mL medium, 3 mL of blood is required). If possible, specimens for culture are obtained before giving antibiotics. Specimens are placed in sterile containers, and sufficient quantities are obtained to allow complete examination. The site of material depends upon the nature of illness and the site where the likelihood of isolation of organism is high. Proper labeling of samples with the patient’s name, age, type of test, date, and site of the collection is an important prerequisite. The specimens for viral cultures are transported in a frozen state at -70 degrees celsius before culture initiation.

Throat and Nasal Swab

These swabs are taken under direct and good visibility and lighting. The preferred sites are areas of exudation, inflammatory membranes, or tonsillar crypts. For nasal specimens, make the patient sit up, bend head slightly backward facing the light source, and then insert and repeatedly rotate the swab into one of the nasal cavities. Spread the isolate on 2 or 3 evenly spread smears. Air-dry the slides and send them to the laboratory for examination.

Sputum Specimen

For certain diseases like mycobacterium tuberculosis (M. tuberculosis), a series of 3 early morning specimens, 8-10 mL in quantity, are collected and stored in a refrigerator. In uncooperative patients, M. tuberculosis can also be recovered from gastric contents by stomach aspiration.

Fecal Specimen

Feces are passed directly into a clean container with a tight cover. They are then transferred to another container. The sample should contain pus, mucus, blood, or any formed elements passed along with stool and include the representative of the first, last, and middle of the stool. Approximately 1 ml (10%) specimen is added to 10 ml alkaline peptone water in diarrhea with cholera suspicion. In viral cases, 1 ml feces are mixed with 9 ml sterile buffered saline and sedimented/centrifuged for half an hour. The supernatant is frozen and stored below – 40 degrees centigrade until further processed.

Physical examination of feces comments on color, odor, consistency, parasites, and pH. Microscopically, we can see food residues, muscle fibers, fats, fatty acid crystals, starch particles, cellulose residues, cells (RBCs, leukocytes, and epithelial), crystals (triple phosphate, calcium oxalate, cholesterol), ova and cysts of parasites, motile amoeba (containing ingested RBC, pseudopodia), mucus, and foreign bodies (hair, wool, etc.).

Urine Specimen

Proper instructions are given to the patients who collect the specimen themselves in a sterile container. An uncontaminated mid-stream urine sample is the best for reporting purposes. Physical examination involves volume, odor, color, appearance, pH, and specific gravity estimation. Chemical examination of urine comments on quantities of normal or abnormal elements such as proteins, glucose, reducing sugars, bile pigments (bilirubin), bile salts, blood, nitrites, ketone bodies, urobilinogen, and Bence Jones proteins, etc. Microscopic examination is an important urinalysis component and can detect ova, parasites, red blood cells, leucocytes, casts, epithelial cells, and crystals. Many other cells can be seen with light-microscopy, like malignant cells, bacteria, yeast cells, trichomonas, filaria, spermatozoa, mucous threads, fat, and other artifacts. Automated equipment like Clinitek 100 and Urilux are used for routine urine examination. They are convenient, accurate, eliminates dependency on visual interpretation, allow compute readings, and decrease the need for re-testing.

Diagnostic Tests

There are hundreds of laboratory tests ordered by physicians that help diagnose, follow-up, and prognosis of various diseases. Herein, we will focus on the most commonly reported groups of tests.

Complete Blood Count (CBC)

Common terms in CBC and their normal values are as follows

Red Blood Cell (RBC) Count: The number of RBCs per volume of blood (normal value: 4.2 to 6.9 x 109/mm3

Hemoglobin (Hb): Amount of oxygen-carrying capacity of blood (normal value: 130 to 180 g/L in males, 120 to 160 g/L in females)

Hematocrit (Hct): Percentage of whole blood occupied by packed RBCs (normal value: 45% to 62% in males, 37% to 48% in females)

Mean Corpuscular Volume (MCV): The measure of RBC size (normal value: 80 to 100 micromillimeter)

Mean Corpuscular Hemoglobin (MCH): Amount of oxygen-carrying hemoglobin inside RBCs (normal value: 27 to 32 pg/cell)

Mean Corpuscular Hemoglobin Concentration (MCHC): Average concentration of Hb inside RBCs (normal value: 32% to 36%)

White Blood Cell (WBC) Count: The number of WBCs per volume of blood (normal value: 4.3 to 11 x 109/mm3)

WBC differential:

• Neutrophils (normal value: 1.8 to 7.8 x 103/mm^3)
• Lymphocytes (normal value: 0.7 to 4.5 x 103/mm^3)
• Monocytes (normal value: 0.1 to 1.0 x 103/mm^3)
• Eosinophils (normal value: 0 to 0.4 x103/mm^3)
• Basophils (normal value: 0 to 0.2 x 103/mm^3)

Platelet Count: The number of platelets per volume of blood (normal value: 150 to 400 x 109/mm^3)

Reticulocytes: The number of immature RBCs in circulating blood (normal value: 1% of total RBC count)

Peripheral Smear

Peripheral smear gives information regarding abnormality in size, shape, color, counts, and composition (inclusions) of cells compared to normal.

Coagulation Profile

Commonly used tests for hemostasis are as follows.

Platelet Count: it gives an account of the number of platelets in the volume of blood (normal value: 150 to 400 x109/L). Low platelet count occurs in thrombocytopenia. It could be primary (isolated thrombocytopenia) without any other underlying cause, or it could be secondary with associated conditions such as HIV, HCV, SLE, CLL. Certain drugs cause drug-induced thrombocytopenia, e.g., aspirin, ethanol, and NSAIDs.

Prothrombin Time (PT): It is also reported along with laboratory control (normal range: 11 to 24s). It measures the extrinsic pathway (factor VII) and common pathway. It is prolonged in vitamin K deficiency, vitamin K antagonist therapy (warfarin), and factor VII deficiency.

International Normalized Ratio (INR): Normal range is 0.9 to 1.2. It is used to monitor warfarin therapy and for the assessment of hepatic function.

Activated Partial Thromboplastin Time (aPTT): It is reported against a normal control (normal range: 22 to 35s). It measures the activity of the common pathway and intrinsic pathway (factors VIII, IX, XI, XII). It is also used to monitor heparin therapy. It is prolonged in hemophilia A (Factor VIII deficiency) and B (Factor IX deficiency).

Other Tests

• Fibrinogen
• D-Dimers
• Specific factor assays (Factor VIII)
• Lupus anticoagulant
• Tests for thrombophilias (activated protein C resistance)
• Von Willebrand tests (vWF antigen, Ristocetin cofactor activity, factor VIII)

Diagnostic Tests in Diabetes Mellitus

• Fasting Blood Glucose (FBG): normal range is <126 mg/dL
• Random Blood Glucose (RBG): normal range is <200 mg/dL
• Glycosylated Hemoglobin (HbA1c): normal value is <6.5 %
• 2 hour 75 gm Oral Glucose Tolerance Test (OGTT): normal value is < 200 mg/dL

Diagnostic Tests in Hepatobiliary Disease

The most commonly ordered tests to evaluate liver function and detect hepatocytes injury are as follows:

• PT/INR: normal range 0.9 to 1.2
• Serum Albumin: normal range 3.5 to 5.0 g/dL
• Serum Bilirubin (total): normal range 0.1 to 1.0 mg/dL
• Serum Bilirubin (direct): normal range 0 to 0.3 mg/dL
• Alanine aminotransferase (ALT): 8 to 20 U/L
• Aspartate aminotransferase (AST): 8 to 20 U/L
• Alkaline phosphatase (ALP): 35 to 100 U/L
• Gamma-glutamyl transferase (GGT): 0 to 30 U/L

Testing Procedures

Microbiological work requires culture media and containers free of all contaminating microorganisms. There are two methods used to kill microorganisms in a sample.

Sterilization

Sterilization is defined as removing all organisms, such as viruses, bacteria, bacterial spores, fungi, and fungal spores, be it pathogenic or non-pathogenic in nature. It is used for culture media, suspended fluids, chemical reagents, containers, and other laboratory equipment.

Methods of Sterilisation

The methods used to achieve an absolute germ-free state are heat (dry heat and moist heat), ionizing radiation (beta electrons and gamma photons irradiation), filtration, and chemical disinfection (gases: ethylene oxide and liquids: glutaraldehydes).

Autoclave

Articles in an autoclave are exposed to moist heat (steam) at temperatures higher than 100 degrees centigrade (mostly 121 degrees centigrade) and pressure greater than the atmospheric pressure. This causes denaturation of protein, destroying the bacterial endospores as well as vegetative cells. Autoclaving is an important method to sterilize most bacteriological culture media, linen, and surgical instruments. The efficiency of an autoclave is checked with chemical and biological controls. The chemical controls include Browne's tube and Bowie Dick tape. The spores of bacillus stereothermophillus are employed as biological control and tested for viability after autoclaving.

Filtration

Filtration is another method of sterilization and is used for attaining bacteria-free solutions and fluids. Filtration is used for materials that are heat-sensitive such as antisera and toxins. There are two types of filtration methods.

1. Surface filtration
2. Depth filtration

In surface filtration, a medium with pores (filter papers, membranes, sieves) is used, and liquids are run through it. Particles that are bigger than the pores' size are retained on the surface of the medium, and the remaining filtrate is collected at the bottom.

Depth filtration follows a similar mechanism as surface filtration, but the particles are retained on the surface and the body of the filter. The fibers network is arranged in such a manner that they retain bi particles. Examples include earthenware (Berkfield and Chamberland), asbestos (Seitz), sintered glass, and cellulose membrane.

Disinfection

Disinfection implies killing most vegetative forms of bacteria, viruses, fungi, and parasites but does not completely eradicate spores and non-vegetative forms. They kill by destroying proteins, lipids, or nucleic acids in the cell itself or its cell membrane. They are particularly used to disinfect surfaces that contact body fluids, tissues, pathological tissue, or samples and microbiological cultures. They are mainly classified into antiseptics and disinfectants.

Antiseptics are diluted forms of disinfectants and are non-toxic for living tissue. Examples include spirit, povidone, alcohol, etc.

Disinfectants are concentrated forms of chemicals and are corrosive to living tissues. They disinfect non-living objects. Examples include phenolic compounds (phenol, Lysol), halogen compounds (chlorine), metallic salts (mercuric chloride, silver nitrate 1%), formaldehyde, volatile solvents (ethyl alcohol, acetone), gaseous disinfectants (gentian violet, potassium permanganate), soaps and detergents. 

Titration

It is a procedure used to identify the quantity of acid or base in a solution with an indicator's help. It helps find an estimate of acids and bases in body fluids such as gastric juice HCl.

Electrophoresis

It is based on the mechanism of the mobility of ions in an electric field. Positively charged ions attract o negative electrode (cathode), and negatively charged ions migrate towards a positive electrode (anode). It is used for the identification and quantification of abnormal patterns of proteins in disease processes. It delineates normal and abnormal protein bands, normal and abnormal hemoglobin, quantification of lipoproteins and isoenzymes.

Chromatography

This is a technique that involves separating pure substances from mixtures. It is based on the fact that different substances follow moving solvents at different speeds. There are two main types.

1. Column chromatography
2. Layer chromatography
3. Thin-layer chromatography
4. Liquid chromatography
5. High-performance liquid chromatography

Some of the chromatography applications include identifying, quantifying, and analyzing drugs, drug metabolites, toxic materials, hemoglobin variants, amino acids and carbohydrates, hormones, etc., in a sample.

Molecular Techniques

Molecular biology involves amplifying small genetic material (DNA/RNA) and its detection using electrophoresis, ELISA (enzyme-linked immunosorbent assay), and chemiluminescence. Examples of amplification techniques are as under:

1. Polymerase chain reaction (PCR)
2. Ligase chain reaction (LCR)
3. Nucleic acid-based amplification (NASBA)
4. Strand displacement assays (SDA)

PCR has useful applications in cases of infectious diseases (tuberculosis, hepatitis B and C, EBV, HIV), genetic disorders (thalassemia, inborn errors of metabolism, insulin gene mutations, cystic fibrosis, muscle dystrophies), antenatal diagnosis, oncogenes, medicolegal science, and tissue typing.

Interfering Factors

There are preset protocols on how to approach, order, and evaluate laboratory tests. Clinical reasoning is necessary before ordering particular laboratory tests. The following queries should be answered before ordering laboratory tests:

• Is the test logically consistent with the diagnostic hypothesis in question?
• Would any change in the clinical condition of the patient justify the test or its repetition?
• Can the test outcome change treatment decisions?
• How safe is the test?
• Is there any harmful consequence if the test is not ordered?[3]

Results, Reporting, and Critical Findings

Approach to Interpretation of CBC

To understand complete blood count, it is important to understand the process of hemopoiesis. Hemopoiesis involves forming mature blood cells from immature cells and consists of a series of distinctively recognizable cells. The production of RBCs, WBCs, platelets, and lymphocytes are named erythropoiesis, granulopoiesis, thrombopoiesis, and lymphopoiesis, respectively. There is a balance between the production and destruction of cells, and each cell has a normal range of count in normal human blood.

If all cell lines are decreased, it is called pancytopenia. Pancytopenia occurs in viral infections, malignancies, bone marrow disorders, autoimmune diseases, a side effect of drugs, and after chemotherapy or radiotherapy.

If RBCs and platelets are decreased, the cause is MAHA/TPA

If a single line is affected, then the approach is different, e.g., low RBCs is called anemia, and a specific approach is followed. Similarly, raised WBCs and/or the presence of different immature cells of white blood cell lineage in the blood are referred to as leukemia.

Anemias

A decrease in hemoglobin level or total circulating red cell mass for a specified age and sex of a person is called anemia. Clinical features include fatigue, weakness, shortness of breath, palpitations, dizziness, headache, and decreased exercise tolerance.

 Diagnostic tests for anemias:

 The following initial investigations are performed if anemia is suspected:

• CBC with differential
• Retic count
• Peripheral blood smear

 Morphologically, anemias are classified into three major types

1. Microcytic hypochromic anemia: RBCs are smaller and in size and contain less hemoglobin than normal cells. Examples: Fe deficiency anemia, thalassemia, sideroblastic anemia, and anemia of chronic disease.
2. Macrocytic anemia: RBCs are larger than normal, and the amount of hemoglobin may be normal or below normal. Examples: B12 deficiency anemia, folate deficiency anemia, drugs, alcoholism, liver disease, and hypothyroidism.
3. Normocytic normochromic anemia: RBCs are normal in size, although the hemoglobin concentration in blood is reduced. Examples: acute blood loss, leukemias, bone marrow infiltration, chronic renal failure, and chronic infections.

Leukemias

Leukemia is defined as the uncontrolled proliferation, abnormal maturation, and accumulation of various intermediate cells. These are divided into acute and chronic leukemia based on disease and the maturation of malignant cells in blood and bone marrow.

Acute Myeloid Leukemia (AML)

It is a rapidly progressive hematological malignancy characterized by the failure of myeloid cells to differentiate beyond the blast stage. The classic finding is circulating blast with Auer rods (azurophilic granules) on peripheral blood film.

Chronic Myeloid Leukemia (CML)

It is an increased proliferation of the granulocytic cell line without loss of their differentiation ability. There is a leukoerythroblastic picture on peripheral blood film with a predominance of immature red cells and granulocytes. Diagnostic test for CML is the presence of bcr-abl fusion gene in more than 90% of patients.

Acute Lymphoblastic Leukemia (ALL)

In ALL, the early lymphoid precursor cells proliferate and replace normal progenitor cells in the bone marrow. Leukemic lymphoblasts lack specific morphological features; therefore, diagnosis requires immunophenotyping. Cytogenetics shows Philadelphia chromosome (Ph) in approximately 25% of adult cases.

Chronic Lymphocytic Leukemia (CLL)

It is a clonal malignancy of mature B-cells. CBC detects population of B lymphocytes >5 x 10/L. On the peripheral blood picture, there are small mature lymphocytes and numerous smudge cells.

Approach to Disorders of Hemostasis

In a patient suspected of coagulation disorder, the investigations should be pre-planned. History and physical examination findings are significant and help decide whether the patient has a vascular defect, platelet defect, or a defect in one of the coagulation factors. It also gives clues of the hereditary or acquired nature of the disorder and narrows down the tests performed. The preliminary tests ordered include platelet count, PT, and aPTT. Further course of action is decided based on the results of these tests.

Approach to Suspected Diabetes Mellitus

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, protein, and fat metabolism resulting from defects in insulin secretion, its action, or both. Typical DM symptoms are polyuria, polydipsia, weight loss, polyphagia, blurred vision, and increased susceptibility to infections. There are two major types of DM.

1. Type 1 diabetes mellitus (immune-mediated, idiopathic)
2. Type 2 diabetes mellitus

Any one of the following is diagnostic

1. FBG ≥ 126 gm/dL OR
2. 2 hr OGTT ≥ 200 mg/dL OR
3. RBG ≥ 200mg/dL along with classic symptoms of DM OR
4. HbA1c ≥ 6.5 %

Impaired fasting glucose:

Patients who have FBG between 109 to 126 mg/dL are labeled to have impaired fasting glucose and are then subjected to 2 hr 75 gm OGTT to confirm DM.

Approach to Liver Function Tests

An increase in PT/INR, serum albumin, serum direct bilirubin, and disproportionately high ALT or AST implies hepatocellular dysfunction. ALT is more specific to the liver; AST comes from multiple sources (especially muscles). AST>ALT is suggestive of alcoholic liver disease.

Serum transaminases >1000 signify direct hepatocyte injury, and the differentials include viral hepatitis, drugs/toxins, autoimmune hepatitis, and hepatic ischemia.

When ALP is elevated alone, rule out the bone disease by fractionating ALP. A disproportionately high ALP and GGT are evident of cholestatic liver diseases (e.g., cholelithiasis, cholangiocarcinoma, sclerosing cholangitis).

Clinical Significance

Laboratory tests are beneficial for patients if an illness is detected, and the monitoring is timely and might have negative effects if the patient complaint is not diagnosed and monitored. Some unnecessary consequences include but are not limited to unwanted phlebotomies leading to phlebitis, pain, journeys, delay in important tests, and improper use of patient and technician time.

A campaign named “Choosing Wisely” by the American Board of Internal Medicine in North America advocates for choosing clinical value tests and encourages patients and healthcare workers to mobilize laboratory resources wisely. Tests that are ordered sometimes have major side effects: they are responsible for a <5% increase in healthcare costs but affect 70% of major decisions regarding admission, treatment, and discharge plan. A study defined by clinical guideline’s recommendations demonstrated that repeat testing is unnecessary in about 25 to 30% of cases. Among medical specialties, family practitioners represent the largest group of doctors ordering laboratory tests, followed by the internal medicine department.[5][6][7][1][2]

Quality Control and Lab Safety

There are two types of quality control methods

Internal Quality Control

It ensures the accuracy and precision of all procedures and equipment by daily checking by the laboratory staff. The following tools are utilized.

Levy-Jennings Chart (LJ Chart)

Levy-Jennings chart estimates accuracy and precision. Serial daily measurements of controls are used to calculate the mean values of standard deviation (SD). The values are then plotted on a graph paper representing mean +-1SD, +-2SD, and +-3SD in horizontal lines. Both normal and abnormal values of controls are plotted on the LJ chart and are monitored daily using Westguard rules.

Five Cycle Quality Control Charts

This chart facilitated the recording of daily values by running controls 5 times repeatedly, giving a wide range of values. Horizontal lines are drawn at mean and +-2SD from standard reference. Control is randomly introduced in daily runs, and the mean is calculated at the end of the day. The results are then plotted on the graph. If the numbers keep increasing or decreasing on one side of the mean value, it is denoted as the upward or downward trend.

Moving Two Standard Deviation Charts

These charts comprise four columns. The left-most column is for recording daily changes from mean assigned to a control. The mean value in the control run during a particular day is calculated, and the difference from the assigned mean is deduced. The values are entered in the assigned slots for respective days. At the end of the week, the values are added and divided by two, which gives 2SD reading. The weekly 2SD values should not differ remarkably from the assigned 2SD value of the control.

Youden Plots

These are specially designed to compare results on two controls. One set is plotted on the x-axis and another set on the y-axis. Zero lines are drawn joining crossing points of axes and +-2SD values. It is inferred that 95% of all results fall in 2SD squares on sides of zero lines.

Results on Patient’s Sample

Specific patient’s sample from the previous day is selected and run to check between run precisions. Mean is calculated from patient results within a defined range and should be relatively constant. The values could be plotted in a graph format. Laboratory errors and non-analytical sampling, and transportation errors affect this method.

Cumulative Sum (CUSUM) Charts

It is applied to daily means or control values. The values are graphically plotted such that the SD value of a given day is added or subtracted from previous day results to obtain a CUSUM chart rather than plotting mean values daily. The outcome should be a straight line of cusum values if accuracy remains unchanged.

2. External Quality Control

This ensures quality control by merging the performance of different laboratories. Samples are distributed to participating laboratories, and results are analyzed critically and statistically. These techniques also allow comparison of various methodologies used by different laboratories and infer recommendations for standard methods.

Laboratory Safety Practices

1. Proper handwashing before and after work
2. Use personal protective equipment, e.g., gloves, mask, goggles, gowns, etc., while handling reagents and testing materials.
3. Do not eat, drink, or smoke on laboratory premises
4. Never pipette with mouth
5. The work area should be free of sharps, glassware, and chemicals
6. Read manufacturer instructions before storage of all laboratory equipment and chemicals
7. Properly label all containers in bold letters and always read labels before using any chemicals
8. Put warning signs at hazardous places
9. Learn proper skills before using unfamiliar equipment and unknown techniques
10. Use caution during transferring and mixing chemicals.

Enhancing Healthcare Team Outcomes

It is the responsibility of basic medical science teachers to teach future clinicians how to approach patients, exercise careful clinical thinking, and apply general principles before requesting laboratory tests. This process begins in medical school by teaching the benefits of high-value, cost-effective laboratory testing. Janssens et al. reviewed various studies and found options to halt the unreasonable demand for laboratory tests. Education and awareness play a vital role, particularly in a teaching university hospital with residents under training. However, it has been found to have limited success due to these institutions' short-term educational opportunities.

Strict protocols need to be followed in the light of clinical reasoning, and proper rationale should be supplied for ordering multiple laboratory tests. The size of the population used to be the predictor of overall laboratory test demand previously. However, an increasing number of primary care physicians might reduce this demand due to fewer visits of patients to emergency departments and urgent care centers. This discourages the practice of defensive medicine and also decreases hospital admissions, surgical operations, and emergency department visits.[8][9][10][7]