Three tube test for hematuria

Why is the three tube test for hematuria done?

  • The three tube test is performed to determine the location of bleeding in the urinary tract.
  • The three tube test also known as the three container method.

How the three tube test is done?

The three tube test is done using three consecutive samples

Three consecutive samples of the urine stream are collected as:

  • 10 to 15 mL of the first urine sample
  • 30 to 40 mL of the midstream urine sample
  • 5 to 10 mL of the last urine sample

The triple-phase urine cytology, or three-tube test, is a diagnostic procedure used to determine the cause of hematuria (blood in the urine). To identify whether the blood is coming from the lower urinary tract (bladder and urethra) or the upper urinary tract (kidneys and ureters), urine samples must be taken at various stages of urination. The test assists in distinguishing between hematuria caused by glomerular and non-glomerular sources.

Here’s how the three-tube test is performed:

  1. First Morning Void (FMV) Sample: The patient provides an initial urine sample immediately upon waking up in the morning. This sample is called the first morning void (FMV). It represents the overnight accumulation of urine and is typically more concentrated.
  2. Midstream Sample: The patient is instructed to drink plenty of fluids and then collect a midstream urine sample after discarding the initial portion of urine that flushes out the urethra. The midstream sample helps evaluate the urinary tract from the bladder to the urethra.
  3. Late Void Sample: The patient is instructed to hold urine for a few hours after the midstream sample collection and then provide a late void urine sample. This sample represents the accumulated urine after holding for a period of time and helps evaluate the urinary tract from the kidneys to the bladder.

Urine samples are collected and then sent to the lab for examination. Red blood cells (RBCs) and other biological components are detected in the urine by microscopic analysis or other diagnostic methods. To ascertain whether the hematuria is predominantly glomerular—originating from the kidneys—or non-glomerular—originating from the bladder or lower urinary tract—the data are evaluated.

The three-tube test is just one component of a comprehensive evaluation for hematuria. It is often performed along with other diagnostic tests, including urine dipstick analysis, urine culture, imaging studies (such as ultrasound or CT scan), and possibly cystoscopy or other invasive procedures, depending on the suspected cause and clinical context.

It’s important to note that the three-tube test should be performed under the guidance of a healthcare professional. They can interpret the results in the context of your specific symptoms, medical history, and other diagnostic findings to determine the appropriate management and further evaluation if needed.

What is the procedure of extracting three consecutive samples

  • Initially the bladder is emptied by passing urine into a series of 3 ounce test tubes
  • Then, The contents of the first test tube and the last  test tube are examined; 
  • The first tube contains the washings from the anterior urethra, 
  • the second test tube comprises of the material from the bladder, and the last test tube constitutes the material from the posterior urethra, prostate, and seminal vesicles

How to understand the results of the three tube test?

The results of the three tube test can be interpreted as Initial hematuria, Terminal hematuria

  1. Initial hematuria
  2. Terminal hematuria
  3. Hematuria in all three samples is seen in lesions that may be anywhere above the bladder neck (bladder, ureters, or kidneys).

What causes Hematuria?

Hematuria is caused by the below conditions

  • Excessive calcium in the urine
  • Stones in the Kidney
  • Cancer of the urinary tract.
  • Conditions that are passed from parent to child (inherited conditions).
  • Exercise that requires a lot of energy.

Initial hematuria

Hematuria (or predominance of RBCs on urine microscopy) primarily in the first sample

What does the initial hematuria indicate?

The initial hematuria is suggestive of an anterior urethral source of bleeding

Terminal hematuria

What does the terminal hematuria indicate?

Terminal Hematuria primarily at the end of the urine stream is suggestive of a lesion at the bladder trigone (bladder neck) or a posterior urethra.

Terminal hematuria is defined as passage of clear urine with blood or blood-stained urine right at the end of the urine stream (“blood notes just at the end of the urine flow”).

Total hematuria is defined as blood-stained urine throughout the entire duration of the urine flow with no ability to readily differentiate between urine and blood.

It’s important to consult with a healthcare professional, such as a urologist, for a proper evaluation and diagnosis of hematuria. They can guide you through the appropriate diagnostic tests based on your specific symptoms, medical history, and individual circumstances.

What is Hematuria?

Blood in the urine is Hematuria.

How is Hematuria defined?

Hematuria is defined as the presence of at least 5 red blood cells/HPF in 3 of 3 consecutive centrifuged specimens obtained at least 7 days apart.

The Blood may be visible in the urine, or it may be identified with a test.

What are the types of Hematuria?

Hematuria, the presence of blood in the urine, can be classified into two main types based on its appearance: gross hematuria and microscopic hematuria.

  1. Gross hematuria: Also known as macroscopic hematuria, this type of hematuria is characterized by visible blood in the urine, which gives the urine a pink, red, or cola-like appearance. Gross hematuria can be alarming due to its visible nature, but the amount of blood may not necessarily indicate the severity of the underlying condition.
  2. Microscopic hematuria: Microscopic hematuria refers to the presence of red blood cells in the urine that can only be detected using a microscope. The urine appears normal in color, and the blood may be identified through laboratory analysis. Microscopic hematuria is often incidentally discovered during routine urine tests or through further evaluation of other urinary symptoms.

Hematuria can be intermittent or persistent. Hematuria may be asymptomatic or symptomatic and may be associated with other urinary tract abnormalities. Hematuria is often first encountered by the primary care provider.


The three-glass urine test, also known as the “Three-Tube Test,” is a diagnostic test used to help determine the origin of blood in the urine (hematuria). It aims to differentiate between blood originating from the urethra, bladder, or kidneys. The test involves collecting sequential urine samples from three separate voids in different containers. Here’s how the test is typically conducted:

  1. Collection:
    • First Void (Tube 1): Ask the patient to urinate into the first container. This initial void helps to flush out any contamination in the urethra.
    • Second Void (Tube 2): Ask the patient to stop midstream and switch to the second container. This sample represents the urine from the bladder, which is usually less likely to be contaminated with any residual blood from the urethra.
    • Third Void (Tube 3): After completing the second void, the patient can continue urinating into the third container. This sample may contain any remaining blood that has reached the bladder during the course of urination.
  2. Analysis:
    • The collected samples are then visually examined for their color and any visible blood clots or particles.
    • Microscopic analysis of the samples can also be performed to quantify the number of red blood cells present in each sample. This can help determine if the source of blood is from the lower urinary tract (urethra or bladder) or the upper urinary tract (kidneys).

Interpreting the Results:

  • If the blood is present only in the first void (Tube 1) and not in the subsequent samples, it suggests that the blood is likely from the urethra or lower urinary tract.
  • If the blood is present in the second and third voids (Tubes 2 and 3) but not in the first void (Tube 1), it suggests that the blood is likely from the bladder.
  • If blood is present in all three voids, it could indicate a more complex urinary tract issue and possibly a source from the kidneys or higher up in the urinary system.

It’s important to note that while the three-tube test can provide useful information, it may not definitively diagnose the underlying cause of hematuria. Additional tests, such as imaging studies (ultrasound, CT scan) and further laboratory analysis, may be needed to pinpoint the exact source and cause of the hematuria. If you’re experiencing blood in your urine, it’s crucial to consult a healthcare professional for proper evaluation and diagnosis.


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